Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them

ABSTRACT

In one aspect, the invention is directed to polypeptides having an amylase and/or glucoamylase activity, polynucleotides encoding the polypeptides, and methods for making and using these polynucleotides and polypeptides. In one aspect, the polypeptides of the invention can be used as amylases, for example, alpha amylases, to catalyze the hydrolysis of polysaccharide, oligosaccharide or starch into sugars. In one aspect, the invention provides delayed release compositions comprising an desired ingredient coated by a latex polymer coating. In alternative embodiments, enzymes are used to make biofuels, e.g., ethanol, butanol, propanol, or a gasoline-ethanol mix, including a bioethanol, biopropanol, biobutanol, or a biodiesel, or for any form of fuel or biomass processing.

This application is a divisional patent application of U.S. Ser. No.13/674,199, filed Nov. 12, 2012, now U.S. Pat. No. 10,100,299, issued onOct. 16, 2018; which is a division patent application of U.S. Ser. No.12/520,523, filed Dec. 21, 2007, now U.S. Pat. No. 8,343,747, whichgranted on Jan. 1, 2013; which is a national phase of PCT applicationPCT/US2007/088631, which published as WO 2008/080093, and having aninternational filing date of Dec. 21, 2007; which claims benefit ofProvisional Application Ser. No. 60/892,823, filed Mar. 2, 2007, andalso claims benefit of Provisional Application Ser. No. 60/877,068,filed Dec. 21, 2006

SEQUENCE LISTING

This application includes a nucleotide and amino acid sequence listingthat is being submitted pursuant to the Electronic Filing System-Web(EFS-Web) Legal Framework, under 37 CFR 1.821 through 1.825 as an asASCII text file named “SequenceListing.txt”, created on Sep. 28, 2018;and the size of the file is 302 KB (309,643 bytes). The entire contentof the sequence listing is hereby incorporated by reference intospecification as filed.

TECHNICAL FIELD

This invention relates to molecular and cellular biology andbiochemistry. In one aspect, the invention is directed to polypeptideshaving an amylase and/or glucoamylase activity, polynucleotides encodingthe polypeptides, and methods for making and using these polynucleotidesand polypeptides. In one aspect, the polypeptides of the invention canbe used as the endo-acting amylases (for example, alpha amylases) or asthe exo-acting glucoamylases, e.g., to catalyze the hydrolysis ofpolysaccharides comprising glucose monomers, such as starch (a polymerof glucose monomers joined by 1,4-alpha or 1,6-alpha linkages), intosugars. In one aspect, the invention is directed to polypeptides havingthermostable amylase and/or glucoamylase activity, including alphaamylases activity or a 1,4-alpha-D-glucan glucohydrolase activity. Inone aspect, the polypeptides of the invention can be used as amylases(for example, alpha amylases) or glucoamylases to catalyze thehydrolysis of polysaccharides such as starch into sugars, such asglucose. The invention is also directed to nucleic acid constructs,vectors, and host cells comprising the nucleic acid sequences of theinvention as well as recombinant methods for producing the polypeptidesof the invention. The invention is also directed to the use of amylasesand/or glucoamylases of the invention in polysaccharide (e.g., starch)conversion processes, including production of high fructose corn syrup(HFCS), ethanol, dextrose, and dextrose syrups.

BACKGROUND

Starch is a complex carbohydrate often found in the human diet. Thestructure of starch is glucose polymers linked by alpha-1,4 andalpha-1,6 glucosidic bonds. Commercially, glucoamylases are used tofurther hydrolyze cornstarch, which has already been partiallyhydrolyzed with an alpha-amylase. The most widely utilized glucoamylaseis produced from the fungus Aspergillus niger; one of the problems withthe commercial use of this enzyme is its relatively low thermostability.

In general, starch to fructose processing consists of four steps:liquefaction of granular starch, saccharification of the liquefiedstarch into dextrose, purification, and isomerization to fructose. Theobject of a starch liquefaction process is to convert a concentratedsuspension of starch polymer granules into a solution of soluble shorterchain length dextrins of low viscosity. This step is essential forconvenient handling with standard equipment and for efficient conversionto glucose or other sugars. To liquefy granular starch, it is necessaryto gelatinize the granules by raising the temperature of the granularstarch to over about 72° C. The heating process instantaneously disruptsthe insoluble starch granules to produce a water soluble starchsolution. The solubilized starch solution is then liquefied by amylase.A starch granule is composed of: 69-74% amylopectin, 26-31% amylose,11-14% water, 0.2-0.4% protein, 0.5-0.9% lipid, 0.05-0.1% ash,0.02-0.03% phosphorus, 0.1% pentosan. Approximately 70% of a granule isamorphous and 30% is crystalline.

Staling of baked products (such as bread) has been recognized as aproblem which becomes more serious as more time lies between the momentof preparation of the bread product and the moment of consumption. Theterm staling is used to describe changes undesirable to the consumer inthe properties of the bread product after leaving the oven, such as anincrease of the firmness of the crumb, a decrease of the elasticity ofthe crumb, and changes in the crust, which becomes tough and leathery.The firmness of the bread crumb increases further during storage up to alevel, which is considered as negative. The increase in crumb firmness,which is considered as the most important aspect of staling, isrecognized by the consumer a long time before the bread product hasotherwise become unsuitable for consumption.

There is a need in the industry for new amylases, e.g., acid amylases,useful for various uses including commercial cornstarch liquefactionprocesses or improved manufacturing having new or improved performancecharacteristics over the industry standard enzymes, e.g., from Bacilluslicheniformis. There is also an industry drive to identify amylases andglucoamylases capable of efficiently hydrolyzing granular starch (e.g.raw granular starch) at low temperatures without the need for a hightemperature starch gelatinization step; the enzymes of the invention,e.g. amylases, glucoamylases and glucosidases, can be utilized tofulfill this need.

There is also a need for new amylases having utility in automatic dishwash (ADW) products and laundry detergent. In ADW products, the amylasewill function at pH 10-11 and at 45-60° C. in the presence of calciumchelators and oxidative conditions. For laundry, activity at pH 9-10 and40° C. in the appropriate detergent matrix will be required. Amylasesare also useful in textile desizing, brewing processes, starchmodification in the paper and pulp industry and other processesdescribed in the art.

SUMMARY

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid sequence having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto a nucleic acid of the invention, e.g., an exemplary nucleic acid ofthe invention, over a region of at least about 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more,residues. In one aspect, the nucleic acid encodes at least onepolypeptide having an amylase and/or glucoamylase activity, and thesequence identities are determined by analysis with a sequencecomparison algorithm or by a visual inspection. In another aspect, theinvention provides nucleic acids for use as probes, inhibitory molecules(e.g., antisense, iRNAs, such as siRNA, microRNA or miRNAs),transcriptional or translational regulation, and the like.

Exemplary nucleic acids of the invention include isolated, synthetic orrecombinant nucleic acids comprising a nucleic acid sequence as setforth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:81 and/or SEQ ID NO:82, and/or subsequences thereof,e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500 or more residues in length, or over the full length of a gene ortranscript (message).

Exemplary nucleic acids of the invention also include isolated,synthetic or recombinant nucleic acids encoding a polypeptide of theinvention, e.g., an exemplary polypeptide having a sequence as set forthin SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76 and/or SEQ ID NO:78, and subsequences thereofand variants thereof, and polypeptides having at least about 50%, 51%,etc., or more, to 100%, as described herein, sequence identity to anexemplary polypeptide of the invention.

In one aspect, the polypeptide of the invention has an endo-actingamylase activity (e.g., as an alpha amylase) or an exo-actingglucoamylase activity (alternative amylase activities described further,below). In one aspect the polypeptide of the invention acts as animmunogen or epitope. In one embodiment, the polypeptides of theinvention can catalyze the hydrolysis of polysaccharides and/oroligosaccharides comprising glucose monomers, such as starch (a polymerof glucose monomers joined by 1,4-alpha or 1,6-alpha linkages).

Amylases and/or glucoamylases of the invention can be used commerciallyin the initial stages (liquefaction) of polysaccharide, oligosaccharideor starch processing; in wet corn milling; in alcohol production; ascleaning agents in detergent matrices; in the textile industry forstarch desizing; in baking applications; in the beverage industry; inoilfields in drilling processes; in inking of recycled paper and inanimal feed. Amylases and/or glucoamylases of the invention can be usedin textile desizing, brewing processes, polysaccharide, oligosaccharideor starch modification in the paper and pulp industry and otherprocesses. For example, the invention provides methods for liquefactionsaccharification as illustrated in FIG. 5 using polypeptides of theinvention.

Amylases and/or glucoamylases of the invention can be used to catalyzethe hydrolysis of polysaccharides, e.g., starches, into sugars; or tohydrolyze internal alpha-1,4-glucosidic linkages in starch to producesmaller molecular weight malto-dextrins. Because the breakdown ofpolysaccharides and/or oligosaccharides, e.g., starches, is important inthe digestive system and in commercial preparation processes, amylasesand/or glucoamylases of the invention are used in foods and feeds andprocesses for preparing them, and in or as digestive aids. Amylasesand/or glucoamylases of the invention can be used in the initial stages(liquefaction) of starch processing; in wet corn milling; in alcoholproduction; as cleaning agents in detergent matrices; in the textileindustry for starch desizing; in baking applications; in the beverageindustry; in oilfields in drilling processes; in inking of recycledpaper; and in animal feed.

Enzymes of the invention can have an exo-acting glucoamylase activityand can be used to further hydrolyze cornstarch which has already beenpartially hydrolyzed with an alpha-amylase (which also can be apolypeptide of the invention) to produce glucose; and in aspect of thisprocess of the invention the glucose is converted to a mixture ofglucose and fructose by a glucose isomerase enzyme. In another aspect ofthis process of the invention, this mixture is enriched with fructose toproduce a high fructose corn syrup. In alternative aspects, polypeptidesof the invention are used in any of step of polysaccharide,oligosaccharide or starch to fructose processing, e.g., including thefour steps: liquefaction of granular starch, saccharification of theliquefied polysaccharide, oligosaccharide or starch into dextrose,purification, and isomerization to fructose. One aspect of the inventionusing at least one polypeptide of the invention comprises apolysaccharide, oligosaccharide or starch liquefaction process forconverting a concentrated suspension of polysaccharide, oligosaccharideor starch polymer granules into a solution of soluble shorter chainlength dextrins of low viscosity.

The invention also provides an enzymatic liquefaction process using atleast one polypeptide of the invention comprising adjusting the pH of agranular polysaccharide, oligosaccharide or starch slurry to the pHoptimum of an enzyme of the invention (e.g., a glucoamylase or anamylase of the invention) to be utilized, e.g., between 6.0 and 6.5, orbetween about 5.5 and 7.0; calcium hydroxide, sodium hydroxide or sodiumcarbonate can be added for this purpose (the addition of calciumhydroxide has the advantage of also providing calcium ions which areknown to stabilize the alpha-amylase against inactivation). In oneaspect, upon addition of the amylase (e.g., an alpha-amylase) of theinvention, the suspension is pumped through a steam jet toinstantaneously raise the temperature to between 80° C. to 115° C., andthe starch is immediately gelatinized and, due to the presence ofalpha-amylase, depolymerized through random hydrolysis of a (1-4)glycosidic bonds by the alpha-amylase to a fluid mass which is easilypumped.

In alternative aspects to this polysaccharide and/or oligosaccharide(e.g., starch) liquefaction process, an amylase (e.g., an alpha-amylase)and/or a glucoamylase of the invention is added to the polysaccharide(e.g., starch) suspension, the suspension is held at a temperature of80-100° C. to partially hydrolyze the granules, e.g., starch granules,and the partially hydrolyzed polysaccharide/starch suspension is pumpedthrough a jet at temperatures in excess of about 105° C. to thoroughlygelatinize any remaining granular structure. After cooling thegelatinized polysaccharide/starch, a second addition of a glucoamylaseand/or an amylase of the invention (e.g., an alpha-amylase) can be madeto further hydrolyze the polysaccharide/starch.

In one aspect, the invention also provides amylase-encoding andglucoamylase-encoding nucleic acids with a common novelty in that theyare derived from mixed cultures, e.g., from environmental sources. Theinvention provides amylase-encoding and glucoamylase-encoding nucleicacids isolated from mixed cultures comprising a nucleic acid sequencehaving at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to an exemplary nucleic acid of theinvention over a region of at least about 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues,wherein the nucleic acid encodes at least one polypeptide having anamylase and/or glucoamylase activity, and the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

In one aspect, the invention provides amylase-encoding andglucoamylase-encoding nucleic acids isolated from mixed cultures, e.g.,from environmental sources, comprising a nucleic acid of the invention,e.g., an exemplary nucleic acid of the invention, e.g., a sequence asset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ IDNO:11, etc., and subsequences thereof, e.g., at least about 10, 15, 20,25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150,1200, 1250, 1300, 1350, 1400, 1450, 1500 or more residues in length, orover the full length of a gene or transcript; or, a nucleic acidencoding a polypeptide of the invention.

In one aspect, the invention also provides amylase-encoding andglucoamylase-encoding nucleic acids with a common novelty in that theyare derived from environmental sources (see Table 1 below, 2^(nd) columnfor examples of sequences isolated from “unknown” or environmentalsources), e.g., mixed environmental sources. In one aspect, theinvention provides amylase-encoding and glucoamylase-encoding nucleicacids isolated from environmental sources, e.g., mixed environmentalsources, comprising a nucleic acid sequence having at least about 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity to an exemplary nucleic acid of the invention over a region ofat least about 25, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550 or more, residues,wherein the nucleic acid encodes at least one polypeptide having anamylase and/or glucoamylase activity, and the sequence identities aredetermined by analysis with a sequence comparison algorithm or by avisual inspection.

In one aspect, the invention provides amylase-encoding andglucoamylase-encoding nucleic acids isolated from environmental sources,e.g., mixed environmental sources, comprising a nucleic acid of theinvention, e.g., an exemplary nucleic acid sequence of the invention asset forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ IDNO:11, etc., SEQ ID NO:583, SEQ ID NO:585, and subsequences thereof,e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500 or more residues in length, or over the full length of a gene ortranscript; or, a nucleic acid encoding a polypeptide of the invention.

In one aspect, the invention also provides glucoamylases and amylases,and amylase-encoding and glucoamylase-encoding nucleic acids, with acommon novelty in that they are derived from archael sources, includingthe archael-derived amylases and/or glucoamylases of the invention.

In one aspect, the sequence comparison algorithm is a BLAST version2.2.2 algorithm where a filtering setting is set to blastall-p blastp-d“nr pataa”-F F, and all other options are set to default.

Another aspect of the invention is an isolated, synthetic or recombinantnucleic acid including at least 10 consecutive bases of a nucleic acidsequence of the invention, sequences substantially identical thereto,and the sequences complementary thereto.

In one aspect, the amylase activity comprises alpha-amylase or aβ-amylase activity, including the ability to hydrolyze internalalpha-1,4-glucosidic linkages in starch to produce smaller molecularweight malto-dextrins. In one aspect, the alpha-amylase activityincludes hydrolyzing internal alpha-1,4-glucosidic linkages in starch atrandom. The glucoamylase and/or amylase activity can comprise analpha-amylase activity, a β-amylase activity, a 1,4-alpha-D-glucanglucohydrolase activity, an exoamylase activity, a glucanalpha-maltotetrahydrolase activity, a maltase activity, an isomaltaseactivity, a glucan 1, 4, alpha-glucosidase activity, analpha-glucosidase activity, a sucrase activity or an agarase activity(e.g., a β-agarase activity).

The amylase activity can comprise hydrolyzing glucosidic bonds. In oneaspect, the glucosidic bonds comprise an alpha-1,4-glucosidic bond. Inanother aspect, the glucosidic bonds comprise an alpha-1,6-glucosidicbond. In one aspect, the amylase activity comprises hydrolyzingglucosidic bonds in polysaccharides, e.g., starches, e.g., liquefiedstarch. The amylase activity can further comprise hydrolyzing glucosidicbonds into maltodextrins. In one aspect, the amylase activity comprisescleaving a maltose or a D-glucose unit from non-reducing end of thestarch.

In one aspect, the isolated, synthetic or recombinant nucleic acidencodes a polypeptide having an amylase and/or glucoamylase activitywhich is thermostable. The polypeptide can retain an amylase activityunder conditions comprising a temperature range of anywhere betweenabout 0° C. to about 37° C., or, between about 37° C. to about 95° C. ormore, e.g., 98° C., 100° C. or more; between about 55° C. to about 85°C., between about 70° C. to about 95° C., or, between about 90° C. toabout 95° C. For example, the exemplary polypeptide having a sequence asset forth in SEQ ID NO:437 is thermostable, retaining 50% activity after25 minutes at 100° C. in the absence of added calcium.

In another aspect, the isolated, synthetic or recombinant nucleic acidencodes a polypeptide having an amylase and/or glucoamylase activitywhich is thermotolerant. The polypeptide can retain an amylase and/orglucoamylase activity after exposure to a temperature in the range fromgreater than 37° C. to about 95° C. or anywhere in the range fromgreater than 55° C. to about 85° C. In one aspect, the polypeptideretains an amylase and/or glucoamylase activity after exposure to atemperature in the range from greater than 90° C. to about 95° C. at pH4.5.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a sequence that hybridizes under stringent conditions to anucleic acid of the invention, e.g., an exemplary nucleic acid of theinvention, a nucleic acid comprising a sequence as set forth in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43,SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53,SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63,SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73,SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81and/or SEQ ID NO:82, and/or fragments or subsequences thereof. In oneaspect, the nucleic acid encodes a polypeptide having an amylase and/orglucoamylase activity. The nucleic acid can be at least about 25, 30,40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500 or more residues in length or the full length ofthe gene or transcript. In one aspect, the stringent conditions includea wash step comprising a wash in 0.2×SSC at a temperature of about 65°C. for about 15 minutes. In one aspect, stringent conditions comprisehybridization under conditions comprising a buffer having 0.15M NaCl for15 minutes at 72° C.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an amylase and/or glucoamylaseactivity, wherein the probe comprises at least about 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000 or more, consecutive bases of a sequence comprising a sequence ofthe invention, or fragments or subsequences thereof, wherein the probeidentifies the nucleic acid by binding or hybridization. The probe cancomprise an oligonucleotide comprising at least about 10 to 50, about 20to 60, about 30 to 70, about 40 to 80, or about 60 to 100 consecutivebases of a sequence comprising a sequence of the invention, or fragmentsor subsequences thereof.

The invention provides a nucleic acid probe for identifying a nucleicacid encoding a polypeptide having an amylase and/or glucoamylaseactivity, wherein the probe comprises a nucleic acid comprising asequence at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more residueshaving at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity to a nucleic acid of the invention,wherein the sequence identities are determined by analysis with asequence comparison algorithm or by visual inspection.

The probe can comprise an oligonucleotide comprising at least about 10to 50, about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to100 consecutive bases of a nucleic acid sequence of the invention, or asubsequence thereof.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having an amylaseand/or glucoamylase activity, wherein the primer pair is capable ofamplifying a nucleic acid comprising a sequence of the invention, orfragments or subsequences thereof. One or each member of theamplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of the sequence.

The invention provides methods of amplifying a nucleic acid encoding apolypeptide having an amylase and/or glucoamylase activity comprisingamplification of a template nucleic acid with an amplification primersequence pair capable of amplifying a nucleic acid sequence of theinvention, or fragments or subsequences thereof.

The invention provides expression cassettes (including, e.g., vectorsand cloning vehicles) comprising a nucleic acid of the invention or asubsequence thereof. In one aspect, the expression cassette can comprisethe nucleic acid that is operably linked to a promoter. The promoter canbe a viral, bacterial, mammalian or plant promoter. In one aspect, theplant promoter can be a potato, rice, corn, wheat, tobacco or barleypromoter. The promoter can be a constitutive promoter. The constitutivepromoter can comprise CaMV35S. In another aspect, the promoter can be aninducible promoter. In one aspect, the promoter can be a tissue-specificpromoter or an environmentally regulated or a developmentally regulatedpromoter. Thus, the promoter can be, e.g., a seed-specific, aleaf-specific, a root-specific, a stem-specific or an abscission-inducedpromoter. In one aspect, the expression cassette can further comprise aplant or plant virus expression vector. In alternative embodiments, theplant promoter is a corn seed embryo-specific: globulin promoter (see,e.g., Belanger (1991) Molecular basis for allelic polymorphism of themaize Globulin-1 gene. Genetics 129:863-872); or a corn seedendosperm-specific: γ-zein promoter (see, e.g., Lopes (1995)Identification of two opaque2 modifier loci in Quality Protein Maize.Mol. Gen. Genet. 247: 603-613); or a rice seed endosperm-specific: GTL1promoter (see, e.g., Takaiwa (1991) Analysis of the 5′ flanking regionresponsible for the endosperm-specific expression of a rice glutelinchimeric gene in transgenic tobacco. Plant Mol. Biol. 16:49-58).

The invention provides cloning vehicles comprising an expressioncassette (e.g., a vector) of the invention or a nucleic acid of theinvention. The cloning vehicle can be a viral vector, a plasmid, aphage, a phagemid, a cosmid, a fosmid, a bacteriophage or an artificialchromosome. The viral vector can comprise an adenovirus vector, aretroviral vector or an adeno-associated viral vector. The cloningvehicle can comprise a bacterial artificial chromosome (BAC), a plasmid,a bacteriophage P1-derived vector (PAC), a yeast artificial chromosome(YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cells (or “host cells”) comprising anucleic acid of the invention or an expression cassette (e.g., a vector)of the invention, or a cloning vehicle of the invention. In one aspect,the transformed cell can be a bacterial cell, a mammalian cell, a fungalcell, a yeast cell, an insect cell or a plant cell. In one aspect, theplant cell can be from any plant, for example plants used for forageand/or feed for any animal, including ruminants, or as a source offeedstock to produce energy or fuel. Plants of particular interest mayinclude crop plants and feedstock plants, for example, maize, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,oat, rye, millet, barley, rice, conifers, grasses, e.g., switch grassand Miscanthus, legume crops, e.g., pea, bean and soybean, starchytuber/roots, e.g., potato, sweet potato, cassava, taro, canna, sugarbeet, sugar cane and the like.

The invention provides transgenic non-human animals comprising a nucleicacid of the invention or an expression cassette (e.g., a vector) of theinvention. In one aspect, the animal is a mouse. The invention providescells or cell lines isolated from these transgenic non-human animals.

The invention provides transgenic plants comprising a nucleic acid ofthe invention or an expression cassette (e.g., a vector) of theinvention. The transgenic plant can be any plant, but in one embodimentthe plant would be used for forage and/or feed for any animal or as afeedstock to produce energy or fuel, such as, maize, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, oat, rye,millet, barley, rice, conifers, grasses, e.g., switch grass andMiscanthus, legume crops, e.g., pea, bean and soybean, starchytuber/roots, e.g., potato, sweet potato, cassava, taro, canna, sugarbeet, sugar cane and the like.

The invention provides transgenic seeds comprising a nucleic acid of theinvention or an expression cassette (e.g., a vector) of the invention.The transgenic seed can from any plant, but in one embodiment the plantwould be used for forage and/or feed for any animal or as a feedstock toproduce energy or fuel, such as, maize, alfalfa, sunflower, Brassica,soybean, cotton, safflower, peanut, sorghum, wheat, oat, rye, millet,barley, rice, conifers, grasses, e.g., switch grass and Miscanthus,legume crops, e.g., pea, bean and soybean, starchy tuber/roots, e.g.,potato, sweet potato, cassava, taro, canna, sugar beet, sugar cane andthe like.

Any plant, plant part, plant tissue, plant seed or plant cell may beused for introduction of a nucleotide of the invention, either stably(e.g., as a transgenic plant, or cell or cell line derived therefrom) ortransiently; thus the invention provides plants, plant parts, planttissues, plant seeds and plant cells comprising a nucleic acid and/orpolypeptide of the invention, wherein the plant can be (but is notlimited to) corn (Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B.juncea), including those Brassica species useful as sources of seed oil,such as canola, alfalfa (Medicago sativa), rice (Oryza sativa), rye(Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet(e.g., pearl millet (Pennisetum glaucum), proso millet (Panicummiliaceum), foxtail millet (Setaria italica), finger millet (Eleusinecoracana)), sunflower (Helianthus annuus), safflower (Carthamustinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco(Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachishypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweetpotato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Cofeaspp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrustrees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis),banana (Musa spp.), avocado (Persea americana), fig (Ficus casica),guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta sp., e.g., Beta vulgaris), sugarcane (Saccharum spp.),Andropogoneae (grasses), Chenopodiaceae (flowering plants), oats,barley, vegetables, ornamentals, and conifers; and vegetables, e.g.,tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa),green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas(Lathyrus spp.), and members of the genus Cucumis such as cucumber (C.sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo); andornamentals, including azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), canna (Cannaceae spp.) and chrysanthemum; andconifers that may be used, including, for example, pines such asloblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine(Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine(Pinus radiata), Douglas-fir (Pseudotsuga menziesii); Western hemlock(Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoiasempervirens); true firs such as silver fir (Abies amabilis) and balsamfir (Abies balsamea); and cedars such as Western red cedar (Thujaplicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis); andleguminous plants, including, but are not limited to, beans and peas,where in alternative aspects beans may include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc., and legumes can include, but are notlimited to, Arachis, e.g., peanuts, Vicia, e.g., crown vetch, hairyvetch, adzuki bean, mung bean, and chickpea, Lupinus, e.g., lupine,trifolium, Phaseolus, e.g., common bean and lima bean, Pisum, e.g.,field bean, Melilotus, e.g., clover, Medicago, e.g., alfalfa, Lotus,e.g., trefoil, lens, e.g., lentil, and false indigo; also includingforage and turf grasses, such as alfalfa, switchgrass (Panicumvirgatum), Miscanthus, orchard grass, tall fescue, perennial ryegrass,creeping bent grass, and redtop.

In alternative aspects, plants, plant parts, plant tissues, plant seedsand plant cells comprising a nucleic acid and/or polypeptide of theinvention also include crop plants and plants used to produce energy orfuel (e.g., ethanol or other biofuels, e.g., bioethanols, biopropanols,biobutanols, or biodiesel), for example, corn, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, oat, rye,millet, barley, rice, conifers, grasses, e.g., switch grass andMiscanthus, legume crops, e.g., pea, bean and soybean, starchytuber/roots, e.g., potato, sweet potato, cassava, taro, canna, sugarcane and/or sugar beet and the like.

The invention provides an antisense oligonucleotide comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a nucleic acid of the invention. The invention providesmethods of inhibiting the translation of an amylase message in a cellcomprising administering to the cell or expressing in the cell anantisense oligonucleotide comprising a nucleic acid sequencecomplementary to or capable of hybridizing under stringent conditions toa nucleic acid of the invention.

The invention provides an isolated, synthetic or recombinant polypeptidecomprising an amino acid sequence having at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequence identityto an exemplary polypeptide or peptide of the invention over a region ofat least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550 or more residues, or over the fulllength of the polypeptide, and the sequence identities are determined byanalysis with a sequence comparison algorithm or by a visual inspection.Exemplary polypeptide or peptide sequences of the invention include SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76 and/or SEQ ID NO:78, and/or subsequences thereof andvariants thereof, e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45,50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500 or more residues in length, or over the full length ofan enzyme. Exemplary polypeptide or peptide sequences of the inventioninclude sequence encoded by a nucleic acid of the invention. Exemplarypolypeptide or peptide sequences of the invention include polypeptidesor peptides specifically bound by an antibody of the invention. In oneaspect, a polypeptide of the invention has at least one amylaseactivity, e.g., an alpha amylase activity.

In alternative embodiments, the polypeptides of the invention lack asignal sequence (leader sequence) and/or a carbohydrate binding module.In alternative embodiments, the polypeptides of the invention furthercomprise one or more heterologous sequences, which can comprise aheterologous signal sequence (leader sequence), a heterologous catalyticdomain (CD) (i.e., active site), or a heterologous carbohydrate bindingmodule, or epitope, purification tag or label. In one aspect, theheterologous signal sequence, heterologous carbohydrate binding moduleor heterologous catalytic domain (CD) is derived from another amylaseenzyme (an amylase other than an enzyme of this invention), or isderived from a non-amylase enzyme.

In alternative embodiments, the polypeptides of the invention have anamylase activity or can be used to generate antibodies which bindspecifically to an exemplary polypeptide of the invention (e.g., SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76 and/or SEQ ID NO:78).

In alternative embodiments, the polypeptides of the invention can besynthetic or in a peptidomimetic form.

Another aspect of the invention is an isolated, synthetic or recombinantpolypeptide or peptide including at least 10, 15, 20, 25, 30, 35, 40,45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450 or 500or more consecutive amino acid residues of a polypeptide or peptidesequence of the invention, sequences substantially identical thereto,and the sequences complementary thereto.

In one aspect, the amylase activity of a polypeptide or peptide of theinvention comprises an alpha-amylase activity, including the ability tohydrolyze internal alpha-1,4-glucosidic linkages in starch to producesmaller molecular weight malto-dextrins. In one aspect, thealpha-amylase activity includes hydrolyzing internalalpha-1,4-glucosidic linkages in starch at random. The amylase activitycan comprise a glucoamylase activity, a 1,4-alpha-D-glucanglucohydrolase activity, an alpha-amylase activity, an exoamylaseactivity, or a β-amylase activity. The amylase activity can comprisehydrolyzing glucosidic bonds. In one aspect, the glucosidic bondscomprise an alpha-1,4-glucosidic bond. In another aspect, the glucosidicbonds comprise an alpha-1,6-glucosidic bond. In one aspect, the amylaseactivity comprises hydrolyzing glucosidic bonds in starch, e.g.,liquefied starch. The amylase activity can further comprise hydrolyzingglucosidic bonds into maltodextrins. In one aspect, the amylase activitycomprises cleaving a maltose or a D-glucose unit from non-reducing endof the starch.

In one aspect, the amylase activity of the invention comprises aglucoamylase activity, which can comprise catalysis of the hydrolysis ofglucosidic bonds. The glucoamylase activity of the invention cancomprise catalyzing the step-wise hydrolytic release of D-glucose fromthe non-reducing ends of starch or other related dextrins. Theglucoamylase activity can comprise a 1,4-alpha-D-glucan glucohydralaseactivity. The glucoamylase activity can comprise catalysis of thehydrolysis of malto-dextrins resulting in the generation of freeglucose. The glucoamylase activity can comprise an exoamylase activity.The glucoamylase activity can comprise an alpha-amylase or a β-amylaseactivity. The hydrolyzed glucosidic bonds can comprisealpha-1,4-glucosidic bonds or alpha-1,6-glucosidic bonds. Theglucoamylase activity can comprise hydrolyzing glucosidic bonds in astarch. The glucoamylase activity can further comprise hydrolyzingglucosidic bonds in the starch to produce maltodextrines. Theglucoamylase activity can comprise cleaving a maltose or a D-glucoseunit from non-reducing end of the starch.

In one aspect, the amylase and/or glucoamylase activity can bethermostable. The polypeptide can retain an amylase and/or glucoamylaseactivity under conditions comprising a temperature range of betweenabout 37° C. to about 95° C., between about 55° C. to about 85° C.,between about 70° C. to about 95° C., or between about 90° C. to about95° C. In another aspect, the amylase and/or glucoamylase activity canbe thermotolerant. The polypeptide can retain an amylase and/orglucoamylase activity after exposure to a temperature in the range fromgreater than 37° C. to about 95° C., or in the range from greater than55° C. to about 85° C. In one aspect, the polypeptide can retain anamylase and/or glucoamylase activity after exposure to a temperature inthe range from greater than 90° C. to about 95° C. at pH 4.5.

In one aspect, the amylase activity and/or glucoamylase activity isthermostable, e.g., wherein the polypeptide retains an amylase activityand/or glucoamylase activity under conditions comprising a temperaturerange from about −100° C. to about −80° C., about −80° C. to about −40°C., about −40° C. to about −20° C., about −20° C. to about 0° C., about0° C. to about 5° C., about 5° C. to about 15° C., about 15° C. to about25° C., about 25° C. to about 37° C., about 37° C. to about 45° C.,about 45° C. to about 55° C., about 55° C. to about 70° C., about 70° C.to about 75° C., about 75° C. to about 85° C., about 85° C. to about 90°C., about 90° C. to about 95° C., about 95° C. to about 100° C., about100° C. to about 105° C., about 105° C. to about 110° C., about 110° C.to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99° C., 100° C.,101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107° C., 108° C.,109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115° C. or more.In some embodiments, the thermostable polypeptides according to theinvention retains activity, e.g., an amylase activity and/orglucoamylase activity, at a temperature in the ranges described above,at about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0,about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5,about pH 8.0, about pH 8.5, about pH 9.0, about pH 9.5, about pH 10.0,about pH 10.5, about pH 11.0, about pH 11.5, about pH 12.0 or more.

In one aspect, the amylase activity and/or glucoamylase activity isthermotolerant, e.g., wherein the polypeptide retains an amylaseactivity and/or glucoamylase activity after exposure to a temperature inthe range from about −100° C. to about −80° C., about −80° C. to about−40° C., about −40° C. to about −20° C., about −20° C. to about 0° C.,about 0° C. to about 5° C., about 5° C. to about 15° C., about 15° C. toabout 25° C., about 25° C. to about 37° C., about 37° C. to about 45°C., about 45° C. to about 55° C., about 55° C. to about 70° C., about70° C. to about 75° C., about 75° C. to about 85° C., about 85° C. toabout 90° C., about 90° C. to about 95° C., about 95° C. to about 100°C., about 100° C. to about 105° C., about 105° C. to about 110° C.,about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99°C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C. 106° C., 107°C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115°C. or more. The thermotolerant polypeptides according to the inventioncan retain activity, e.g. an amylase activity and/or glucoamylaseactivity, after exposure to a temperature in the range from about −100°C. to about −80° C., about −80° C. to about −40° C., about −40° C. toabout −20° C., about −20° C. to about 0° C., about 0° C. to about 5° C.,about 5° C. to about 15° C., about 15° C. to about 25° C., about 25° C.to about 37° C., about 37° C. to about 45° C., about 45° C. to about 55°C., about 55° C. to about 70° C., about 70° C. to about 75° C., about75° C. to about 85° C., about 85° C. to about 90° C., about 90° C. toabout 95° C., about 95° C. to about 100° C., about 100° C. to about 105°C., about 105° C. to about 110° C., about 110° C. to about 120° C., or95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103°C., 104° C., 105° C., 106° C., 107° C., 108° C., 109° C., 110° C., 111°C., 112° C., 113° C., 114° C., 115° C. or more. In some embodiments, thethermotolerant polypeptides according to the invention retains activity,e.g. amylase activity and/or glucoamylase activity, after exposure to atemperature in the ranges described above, at about pH 3.0, about pH3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH11.0, about pH 11.5, about pH 12.0 or more.

In one aspect, the amylase activity and/or glucoamylase activity ofpolypeptides encoded by nucleic acids of the invention retain activityunder acidic conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH4.5, pH 4.0, pH 3.5, pH 3.0 or less (more acidic) pH, or, retain anamylase activity and/or glucoamylase activity after exposure to acidicconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4.0,pH 3.5, pH 3.0 or less (more acidic) pH; or, retain activity under basicconditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (more basic) or,retain an amylase activity and/or glucoamylase activity after exposureto basic conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9,pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (morebasic). In one aspect, amylase activity and/or glucoamylase activity ofpolypeptides encoded by nucleic acids of the invention retain activityat a temperature of at least about 80° C., 81° C., 82° C., 83° C., 84°C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93°C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C.,102° C., 103° C., 103.5° C., 104° C., 105° C., 107° C., 108° C., 109° C.or 110° C., or more, and a basic pH of at least about pH 7.5 pH 8.0, pH8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 ormore (more basic).

In one aspect, the isolated, synthetic or recombinant polypeptide cancomprise the polypeptide of the invention that lacks a signal sequence.In one aspect, the isolated, synthetic or recombinant polypeptide cancomprise the polypeptide of the invention comprising a heterologoussignal sequence, such as a heterologous amylase or non-amylase signalsequence.

In one aspect, the invention provides a signal sequence comprising apeptide as set forth in Table 1. In one aspect, the invention provides asignal sequence consisting of a peptide as set forth in Table 1. In oneaspect, the invention provides chimeric proteins comprising a firstdomain comprising a signal sequence of the invention and at least asecond domain. The protein can be a fusion protein. The second domaincan comprise an enzyme. The enzyme can be any glucoamylase and/oramylase (e.g., a glucoamylase or an amylase of the invention, or,another amylase or glucoamylase).

In one aspect, the enzymatic activity (e.g., glucoamylase and/or anamylase activity) of an enzyme of this invention comprises a specificactivity at about 37° C. in the range from about 1 to about 1200 unitsper milligram of protein, or, about 100 to about 1000 units permilligram of protein. In another aspect, the enzymatic activity (e.g.,glucoamylase and/or an amylase activity) of an enzyme of this inventioncomprises a specific activity from about 100 to about 1000 units permilligram of protein, or, from about 500 to about 750 units permilligram of protein. Alternatively, the enzymatic activity (e.g.,glucoamylase and/or an amylase activity) of an enzyme of this inventioncomprises a specific activity at 37° C. in the range from about 1 toabout 750 units per milligram of protein, or, from about 500 to about1200 units per milligram of protein. In one aspect, the enzymaticactivity (e.g., glucoamylase and/or an amylase activity) of an enzyme ofthis invention comprises a specific activity at 37° C. in the range fromabout 1 to about 500 units per milligram of protein, or, from about 750to about 1000 units per milligram of protein. In another aspect, theenzymatic activity (e.g., glucoamylase and/or an amylase activity) of anenzyme of this invention comprises a specific activity at 37° C. in therange from about 1 to about 250 units per milligram of protein.Alternatively, the enzymatic activity (e.g., glucoamylase and/or anamylase activity) of an enzyme of this invention comprises a specificactivity at 37° C. in the range from about 1 to about 100 units permilligram of protein. In another aspect, the thermotolerance comprisesretention of at least half of the specific activity of the enzymaticactivity (e.g., glucoamylase and/or an amylase activity) of an enzyme ofthis invention at 37° C. after being heated to an elevated temperature,such as a temperature from about 0° C. to about 20° C., about 20° C. toabout 37° C., about 37° C. to about 50° C., about 50° C. to about 70°C., about 70° C. to about 75° C., about 75° C. to about 80° C., about80° C. to about 85° C., about 85° C. to about 90° C., about 90° C. toabout 95° C., about 95° C. to about 100° C., about 100° C. to about 110°C., or higher. Alternatively, the thermotolerance can comprise retentionof specific activity at 37° C. in the range from about 1 to about 1200units per milligram of protein, or, from about 500 to about 1000 unitsper milligram of protein, after being heated to an elevated temperature.In another aspect, the thermotolerance can comprise retention ofspecific activity at 37° C. in the range from about 1 to about 500 unitsper milligram of protein after being heated to an elevated temperature,as described above.

The invention provides isolated, synthetic or recombinant polypeptidesof the invention, wherein the polypeptide comprises at least oneglycosylation site. In one aspect, glycosylation can be an N-linkedglycosylation. In one aspect, the polypeptide can be glycosylated afterbeing expressed in a P. pastoris or a S. pombe. The invention alsoprovides methods for adding glycosylation to a polypeptide, eitherpost-translationally or chemically, to change the property of thepolypeptides, e.g., its thermal stability, solubility, tendency toaggregate, and the like.

In one aspect, the polypeptide can retain the enzymatic activity (e.g.,glucoamylase and/or an amylase activity) of an enzyme of this inventionunder conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH4.0, pH 3.5, pH 3.0 or less (more acidic) pH. In another aspect, thepolypeptide can retain the enzymatic activity (e.g., glucoamylase and/oran amylase activity) of an enzyme of this invention under conditionscomprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH10.5, pH 11.0, pH 11.5, pH 12, pH 12.5 or more (more basic) pH. In oneaspect, the polypeptide can retain the enzymatic activity (e.g.,glucoamylase and/or an amylase activity) of an enzyme of this inventionafter exposure to conditions comprising about pH 6.5, pH 6, pH 5.5, pH5, pH 4.5, pH 4.0, pH 3.5, pH 3.0 or less (more acidic) pH. In anotheraspect, the polypeptide can retain the enzymatic activity (e.g.,glucoamylase and/or an amylase activity) of an enzyme of this inventionafter exposure to conditions comprising about pH 7, pH 7.5 pH 8.0, pH8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11.0, pH 11.5, pH 12, pH 12.5 ormore (more basic) pH.

The invention provides protein preparations comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, asolid or a gel.

The invention provides heterodimers comprising a polypeptide of theinvention and a second domain. In one aspect, the second domain can be apolypeptide and the heterodimer can be a fusion protein. In one aspect,the second domain can be an epitope or a tag. In one aspect, theinvention provides homodimers comprising a polypeptide of the invention.

The invention provides immobilized polypeptides having an amylase and/orglucoamylase activity, wherein the polypeptide comprises a polypeptideof the invention, a polypeptide encoded by a nucleic acid of theinvention, or a polypeptide comprising a polypeptide of the inventionand a second domain. In one aspect, the polypeptide can be immobilizedon a cell, a metal, a resin, a polymer, a ceramic, a glass, amicroelectrode, a graphitic particle, a bead, a gel, a plate, an arrayor a capillary tube.

The invention provides arrays comprising an immobilized nucleic acid ofthe invention. The invention provides arrays comprising an antibody ofthe invention.

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to a polypeptide of the invention or to apolypeptide encoded by a nucleic acid of the invention. The antibody canbe a monoclonal or a polyclonal antibody. The invention provideshybridomas comprising an antibody of the invention, e.g., an antibodythat specifically binds to a polypeptide of the invention or to apolypeptide encoded by a nucleic acid of the invention.

The invention provides food supplements for an animal comprising apolypeptide of the invention, e.g., a polypeptide encoded by the nucleicacid of the invention. In one aspect, the polypeptide in the foodsupplement can be glycosylated. The invention provides edible enzymedelivery matrices comprising a polypeptide of the invention, e.g., apolypeptide encoded by the nucleic acid of the invention. In one aspect,the delivery matrix comprises a pellet. In one aspect, the polypeptidecan be glycosylated. In one aspect, the amylase activity isthermotolerant. In another aspect, the amylase activity is thermostable.

The invention provides method of isolating or identifying a polypeptidehaving an amylase and/or glucoamylase activity comprising the steps of:(a) providing an antibody of the invention; (b) providing a samplecomprising polypeptides; and (c) contacting the sample of step (b) withthe antibody of step (a) under conditions wherein the antibody canspecifically bind to the polypeptide, thereby isolating or identifying apolypeptide having an amylase and/or glucoamylase activity.

The invention provides methods of making an anti-amylase antibodycomprising administering to a non-human animal a nucleic acid of theinvention or a polypeptide of the invention or subsequences thereof inan amount sufficient to generate a humoral immune response, therebymaking an anti-glycoamylase and/or anti-amylase antibody. The inventionprovides methods of making an anti-glycoamylase and/or anti-amylaseimmune comprising administering to a non-human animal a nucleic acid ofthe invention or a polypeptide of the invention or subsequences thereofin an amount sufficient to generate an immune response.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid of the inventionoperably linked to a promoter; and (b) expressing the nucleic acid ofstep (a) under conditions that allow expression of the polypeptide,thereby producing a recombinant polypeptide. In one aspect, the methodcan further comprise transforming a host cell with the nucleic acid ofstep (a) followed by expressing the nucleic acid of step (a), therebyproducing a recombinant polypeptide in a transformed cell.

The invention provides methods for identifying a polypeptide having anamylase and/or glucoamylase activity comprising the following steps: (a)providing a polypeptide of the invention; or a polypeptide encoded by anucleic acid of the invention; (b) providing an amylase substrate; and(c) contacting the polypeptide or a fragment or variant thereof of step(a) with the substrate of step (b) and detecting a decrease in theamount of substrate or an increase in the amount of a reaction product,wherein a decrease in the amount of the substrate or an increase in theamount of the reaction product detects a polypeptide having an amylaseand/or glucoamylase activity. In one aspect, the substrate can be apolysaccharide, oligosaccharide or starch, e.g., a liquefied starch.

The invention provides methods for identifying an amylase orglucoamylase substrate comprising the following steps: (a) providing apolypeptide of the invention; or a polypeptide encoded by a nucleic acidof the invention; (b) providing a test substrate; and (c) contacting thepolypeptide of step (a) with the test substrate of step (b) anddetecting a decrease in the amount of substrate or an increase in theamount of reaction product, wherein a decrease in the amount of thesubstrate or an increase in the amount of a reaction product identifiesthe test substrate as an amylase or a glucoamylase substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising the following steps: (a)expressing a nucleic acid or a vector comprising the nucleic acid underconditions permissive for translation of the nucleic acid to apolypeptide, wherein the nucleic acid comprises a nucleic acid of theinvention, or, providing a polypeptide of the invention; (b) providing atest compound; (c) contacting the polypeptide with the test compound;and (d) determining whether the test compound of step (b) specificallybinds to the polypeptide.

The invention provides methods for identifying a modulator of an amylaseor a glucoamylase activity comprising the following steps: (a) providinga polypeptide of the invention or a polypeptide encoded by a nucleicacid of the invention; (b) providing a test compound; (c) contacting thepolypeptide of step (a) with the test compound of step (b) and measuringan activity of the amylase or glucoamylase, wherein a change in theamylase or glucoamylase activity measured in the presence of the testcompound compared to the activity in the absence of the test compoundprovides a determination that the test compound modulates the amylase orglucoamylase activity. In one aspect, the amylase or a glucoamylaseactivity can be measured by providing an amylase or glucoamylasesubstrate and detecting a decrease in the amount of the substrate or anincrease in the amount of a reaction product, or, an increase in theamount of the substrate or a decrease in the amount of a reactionproduct. A decrease in the amount of the substrate or an increase in theamount of the reaction product with the test compound as compared to theamount of substrate or reaction product without the test compoundidentifies the test compound as an activator of amylase or glucoamylaseactivity. An increase in the amount of the substrate or a decrease inthe amount of the reaction product with the test compound as compared tothe amount of substrate or reaction product without the test compoundidentifies the test compound as an inhibitor of amylase or glucoamylaseactivity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence of the invention(e.g., a polypeptide encoded by a nucleic acid of the invention). In oneaspect, the computer system can further comprise a sequence comparisonalgorithm and a data storage device having at least one referencesequence stored thereon. In another aspect, the sequence comparisonalgorithm comprises a computer program that indicates polymorphisms. Inone aspect, the computer system can further comprise an identifier thatidentifies one or more features in said sequence. The invention providescomputer readable media having stored thereon a polypeptide sequence ora nucleic acid sequence of the invention. The invention provides methodsfor identifying a feature in a sequence comprising the steps of: (a)reading the sequence using a computer program which identifies one ormore features in a sequence, wherein the sequence comprises apolypeptide sequence or a nucleic acid sequence of the invention; and(b) identifying one or more features in the sequence with the computerprogram. The invention provides methods for comparing a first sequenceto a second sequence comprising the steps of: (a) reading the firstsequence and the second sequence through use of a computer program whichcompares sequences, wherein the first sequence comprises a polypeptidesequence or a nucleic acid sequence of the invention; and (b)determining differences between the first sequence and the secondsequence with the computer program. The step of determining differencesbetween the first sequence and the second sequence can further comprisethe step of identifying polymorphisms. In one aspect, the method canfurther comprise an identifier that identifies one or more features in asequence. In another aspect, the method can comprise reading the firstsequence using a computer program and identifying one or more featuresin the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an amylase and/or glucoamylaseactivity from an environmental sample comprising the steps of: (a)providing an amplification primer sequence pair for amplifying a nucleicacid encoding a polypeptide having an amylase and/or glucoamylaseactivity, wherein the primer pair is capable of amplifying a nucleicacid of the invention; (b) isolating a nucleic acid from theenvironmental sample or treating the environmental sample such thatnucleic acid in the sample is accessible for hybridization to theamplification primer pair; and, (c) combining the nucleic acid of step(b) with the amplification primer pair of step (a) and amplifyingnucleic acid from the environmental sample, thereby isolating orrecovering a nucleic acid encoding a polypeptide having an amylaseand/or glucoamylase activity from an environmental sample. One or eachmember of the amplification primer sequence pair can comprise anoligonucleotide comprising at least about 10 to 50 consecutive bases ofa sequence of the invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an amylase and/or glucoamylaseactivity from an environmental sample comprising the steps of: (a)providing a polynucleotide probe comprising a nucleic acid of theinvention or a subsequence thereof; (b) isolating a nucleic acid fromthe environmental sample or treating the environmental sample such thatnucleic acid in the sample is accessible for hybridization to apolynucleotide probe of step (a); (c) combining the isolated nucleicacid or the treated environmental sample of step (b) with thepolynucleotide probe of step (a); and (d) isolating a nucleic acid thatspecifically hybridizes with the polynucleotide probe of step (a),thereby isolating or recovering a nucleic acid encoding a polypeptidehaving an amylase and/or glucoamylase activity from an environmentalsample. The environmental sample can comprise a water sample, a liquidsample, a soil sample, an air sample or a biological sample. In oneaspect, the biological sample can be derived from a bacterial cell, aprotozoan cell, an insect cell, a yeast cell, a plant cell, a fungalcell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having an amylase and/or glucoamylase activitycomprising the steps of: (a) providing a template nucleic acidcomprising a nucleic acid of the invention; and (b) modifying, deletingor adding one or more nucleotides in the template sequence, or acombination thereof, to generate a variant of the template nucleic acid.In one aspect, the method can further comprise expressing the variantnucleic acid to generate a variant amylase or glucoamylase polypeptide.The modifications, additions or deletions can be introduced by a methodcomprising error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR)or a combination thereof. In another aspect, the modifications,additions or deletions are introduced by a method comprisingrecombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation and acombination thereof.

In one aspect, the method can be iteratively repeated until an amylaseor glucoamylase having an altered or different activity or an altered ordifferent stability from that of a polypeptide encoded by the templatenucleic acid is produced. In one aspect, the variant amylase orglucoamylase polypeptide is thermotolerant, and retains some activityafter being exposed to an elevated temperature. In another aspect, thevariant amylase or glucoamylase polypeptide has increased glycosylationas compared to the amylase or glucoamylase encoded by a template nucleicacid. Alternatively, the variant amylase or glucoamylase polypeptide hasan amylase or glucoamylase activity under a high temperature, whereinthe amylase or glucoamylase encoded by the template nucleic acid is notactive under the high temperature. In one aspect, the method can beiteratively repeated until an amylase or glucoamylase coding sequencehaving an altered codon usage from that of the template nucleic acid isproduced. In another aspect, the method can be iteratively repeateduntil an amylase or glucoamylase gene having higher or lower level ofmessage expression or stability from that of the template nucleic acidis produced.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an amylase and/or glucoamylase activity toincrease its expression in a host cell, the method comprising thefollowing steps: (a) providing a nucleic acid of the invention encodinga polypeptide having an amylase and/or glucoamylase activity; and, (b)identifying a non-preferred or a less preferred codon in the nucleicacid of step (a) and replacing it with a preferred or neutrally usedcodon encoding the same amino acid as the replaced codon, wherein apreferred codon is a codon over-represented in coding sequences in genesin the host cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell, therebymodifying the nucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an amylase and/or glucoamylase activity;the method comprising the following steps: (a) providing a nucleic acidof the invention; and, (b) identifying a codon in the nucleic acid ofstep (a) and replacing it with a different codon encoding the same aminoacid as the replaced codon, thereby modifying codons in a nucleic acidencoding an amylase or glucoamylase.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an amylase and/or glucoamylase activity toincrease its expression in a host cell, the method comprising thefollowing steps: (a) providing a nucleic acid of the invention encodingan amylase or glucoamylase polypeptide; and, (b) identifying anon-preferred or a less preferred codon in the nucleic acid of step (a)and replacing it with a preferred or neutrally used codon encoding thesame amino acid as the replaced codon, wherein a preferred codon is acodon over-represented in coding sequences in genes in the host cell anda non-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having an amylase and/or glucoamylase activity todecrease its expression in a host cell, the method comprising thefollowing steps: (a) providing a nucleic acid of the invention; and (b)identifying at least one preferred codon in the nucleic acid of step (a)and replacing it with a non-preferred or less preferred codon encodingthe same amino acid as the replaced codon, wherein a preferred codon isa codon over-represented in coding sequences in genes in a host cell anda non-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified amylase or glucoamylase active sites orsubstrate binding sites, wherein the modified active sites or substratebinding sites are derived from a first nucleic acid comprising asequence encoding a first active site or a first substrate binding sitethe method comprising the following steps: (a) providing a first nucleicacid encoding a first active site or first substrate binding site,wherein the first nucleic acid sequence comprises a sequence thathybridizes under stringent conditions to a nucleic acid of theinvention, and the nucleic acid encodes an amylase or glucoamylaseactive site or an amylase or glucoamylase substrate binding site; (b)providing a set of mutagenic oligonucleotides that encodenaturally-occurring amino acid variants at a plurality of targetedcodons in the first nucleic acid; and, (c) using the set of mutagenicoligonucleotides to generate a set of active site-encoding or substratebinding site-encoding variant nucleic acids encoding a range of aminoacid variations at each amino acid codon that was mutagenized, therebyproducing a library of nucleic acids encoding a plurality of modifiedamylase or glucoamylase active sites or substrate binding sites. In oneaspect, the method comprises mutagenizing the first nucleic acid of step(a) by a method comprising an optimized directed evolution system, genesite-saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),error-prone PCR, shuffling, oligonucleotide-directed mutagenesis,assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassettemutagenesis, recursive ensemble mutagenesis, exponential ensemblemutagenesis, site-specific mutagenesis, gene reassembly, gene sitesaturated mutagenesis (GSSM), synthetic ligation reassembly (SLR) and acombination thereof. In another aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprisingthe following steps: (a) providing a plurality of biosynthetic enzymescapable of synthesizing or modifying a small molecule, wherein one ofthe enzymes comprises an amylase or glucoamylase enzyme encoded by anucleic acid of the invention; (b) providing a substrate for at leastone of the enzymes of step (a); and (c) reacting the substrate of step(b) with the enzymes under conditions that facilitate a plurality ofbiocatalytic reactions to generate a small molecule by a series ofbiocatalytic reactions. The invention provides methods for modifying asmall molecule comprising the following steps: (a) providing an amylaseor glucoamylase enzyme, wherein the enzyme comprises a polypeptide ofthe invention, or, a polypeptide encoded by a nucleic acid of theinvention, or a subsequence thereof; (b) providing a small molecule; and(c) reacting the enzyme of step (a) with the small molecule of step (b)under conditions that facilitate an enzymatic reaction catalyzed by theamylase or glucoamylase enzyme, thereby modifying a small molecule by anamylase or glucoamylase enzymatic reaction. In one aspect, the methodcan comprise a plurality of small molecule substrates for the enzyme ofstep (a), thereby generating a library of modified small moleculesproduced by at least one enzymatic reaction catalyzed by the amylase orglucoamylase enzyme. In one aspect, the method can comprise a pluralityof additional enzymes under conditions that facilitate a plurality ofbiocatalytic reactions by the enzymes to form a library of modifiedsmall molecules produced by the plurality of enzymatic reactions. Inanother aspect, the method can further comprise the step of testing thelibrary to determine if a particular modified small molecule whichexhibits a desired activity is present within the library. The step oftesting the library can further comprise the steps of systematicallyeliminating all but one of the biocatalytic reactions used to produce aportion of the plurality of the modified small molecules within thelibrary by testing the portion of the modified small molecule for thepresence or absence of the particular modified small molecule with adesired activity, and identifying at least one specific biocatalyticreaction that produces the particular modified small molecule of desiredactivity.

The invention provides methods for determining a functional fragment ofan amylase or glucoamylase enzyme comprising the steps of: (a) providingan amylase or glucoamylase enzyme, wherein the enzyme comprises apolypeptide of the invention, or a polypeptide encoded by a nucleic acidof the invention, or a subsequence thereof; and (b) deleting a pluralityof amino acid residues from the sequence of step (a) and testing theremaining subsequence for an amylase or glucoamylase activity, therebydetermining a functional fragment of an amylase or glucoamylase enzyme.In one aspect, the amylase or glucoamylase activity is measured byproviding an amylase or glucoamylase substrate and detecting a decreasein the amount of the substrate or an increase in the amount of areaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising the following steps: (a) making a modified cell bymodifying the genetic composition of a cell, wherein the geneticcomposition is modified by addition to the cell of a nucleic acid of theinvention; (b) culturing the modified cell to generate a plurality ofmodified cells; (c) measuring at least one metabolic parameter of thecell by monitoring the cell culture of step (b) in real time; and, (d)analyzing the data of step (c) to determine if the measured parameterdiffers from a comparable measurement in an unmodified cell undersimilar conditions, thereby identifying an engineered phenotype in thecell using real-time metabolic flux analysis. In one aspect, the geneticcomposition of the cell can be modified by a method comprising deletionof a sequence or modification of a sequence in the cell, or, knockingout the expression of a gene. In one aspect, the method can furthercomprise selecting a cell comprising a newly engineered phenotype. Inanother aspect, the method can comprise culturing the selected cell,thereby generating a new cell strain comprising a newly engineeredphenotype.

The invention provides methods for hydrolyzing polysaccharide,oligosaccharide or starch, comprising the following steps: (a) providinga polypeptide having an amylase and/or glucoamylase activity, whereinthe polypeptide comprises a polypeptide of the invention; (b) providinga composition comprising a polysaccharide, oligosaccharide or starch;and (c) contacting the polypeptide of step (a) with the composition ofstep (b) under conditions wherein the polypeptide hydrolyzes thepolysaccharide, oligosaccharide or starch. In one aspect, thecomposition comprising polysaccharide, oligosaccharide or starch, thatcomprises an alpha-1,4-glucosidic bond or an alpha-1,6-glucosidic bond.In one aspect, the amylase activity is an alpha-amylase or abeta-amylase activity. In one aspect, the alpha-amylase activityhydrolyzes internal bonds in a starch or other polysaccharide.

The invention provides methods for liquefying or removing apolysaccharide, oligosaccharide or starch, from a composition comprisingthe following steps: (a) providing a polypeptide having an amylaseand/or glucoamylase activity, wherein the polypeptide comprises apolypeptide of the invention; (b) providing a composition comprising apolysaccharide, oligosaccharide or starch; and (c) contacting thepolypeptide of step (a) with the composition of step (b) underconditions wherein the polypeptide removes or liquefies thepolysaccharide, oligosaccharide or starch.

The invention provides methods of increasing thermotolerance orthermostability of an amylase polypeptide, the method comprisingglycosylating an amylase or glucoamylase polypeptide, wherein thepolypeptide comprises at least thirty contiguous amino acids of apolypeptide of the invention; or a polypeptide encoded by a nucleic acidsequence of the invention, thereby increasing the thermotolerance orthermostability of the amylase or glucoamylase polypeptide. In oneaspect, the amylase or glucoamylase specific activity can bethermostable or thermotolerant at a temperature in the range fromgreater than about 37° C. to about 95° C.

The invention provides methods for overexpressing a recombinant amylaseor glucoamylase polypeptide in a cell comprising expressing a vectorcomprising a nucleic acid comprising a nucleic acid of the invention ora nucleic acid sequence of the invention, wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by visual inspection, wherein overexpression is effected byuse of a high activity promoter, a dicistronic vector or by geneamplification of the vector.

The invention provides detergent compositions comprising a polypeptideof the invention or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide comprises an amylase or glucoamylaseactivity. In one aspect, the amylase or glucoamylase can be anonsurface-active amylase or glucoamylase. In another aspect, theamylase or glucoamylase can be a surface-active amylase or glucoamylase.

The invention provides methods for washing an object comprising thefollowing steps: (a) providing a composition comprising a polypeptidehaving an amylase and/or glucoamylase activity, wherein the polypeptidecomprises: a polypeptide of the invention or a polypeptide encoded by anucleic acid of the invention; (b) providing an object; and (c)contacting the polypeptide of step (a) and the object of step (b) underconditions wherein the composition can wash the object.

The invention provides methods for hydrolyzing a polysaccharide,oligosaccharide or starch, e.g., in a feed or a food prior toconsumption by an animal, comprising the following steps: (a) obtaininga composition, e.g., a feed material, comprising a polysaccharide,oligosaccharide or starch, wherein the polypeptide comprises: apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention; and (b) adding the polypeptide of step (a) to thecomposition, e.g., the feed or food material, in an amount sufficientfor a sufficient time period to cause hydrolysis of the polysaccharide,oligosaccharide or starch, thereby hydrolyzing the polysaccharide,oligosaccharide or starch. In one aspect, the food or feed comprisesrice, corn, barley, wheat, legumes, or potato.

For example, in one embodiment, the invention provide compositionscomprising a combination of an amylase and a glucoamylase (where one orboth of these enzymes is an enzyme of this invention) for the hydrolysisof a polysaccharide, oligosaccharide or starch, e.g., a rice, corn,barley, wheat, legumes, or potato starch. In one aspect, enzyme loadingfor a combination of an amylase and a glucoamylase comprises a 1:10amylase: glucoamylase ratio; wherein in one embodiment a total enzymeload is between 0.015%-0.0255% enzyme necessary to completely hydrolyze33% of a flour, e.g., a corn flour. Alternative exemplary ranges forloading are around 0.01% (w/w) for hydrolysis of 23% purified cornstarch and between 0.015%-0.2% (w/w) for hydrolysis of corn starch in33% corn flour.

The invention provides methods for textile desizing comprising thefollowing steps: (a) providing a polypeptide having an amylase and/orglucoamylase activity, wherein the polypeptide comprises a polypeptideof the invention or a polypeptide encoded by a nucleic acid of theinvention; (b) providing a fabric; and (c) contacting the polypeptide ofstep (a) and the fabric of step (b) under conditions wherein the amylaseor glucoamylase can desize the fabric.

The invention provides methods for deinking of paper or fiberscomprising the following steps: (a) providing a polypeptide having anamylase and/or glucoamylase activity, wherein the polypeptide comprisesa polypeptide of the invention; (b) providing a composition comprisingpaper or fiber; and (c) contacting the polypeptide of step (a) and thecomposition of step (b) under conditions wherein the polypeptide candeink the paper or fiber.

The invention provides methods for treatment of lignocellulosic fiberscomprising the following steps: (a) providing a polypeptide having anamylase and/or glucoamylase activity, wherein the polypeptide comprisesa polypeptide of the invention; (b) providing a lignocellulosic fiber;and (c) contacting the polypeptide of step (a) and the fiber of step (b)under conditions wherein the polypeptide can treat the fiber therebyimproving the fiber properties.

The invention provides methods for producing a high-maltose or ahigh-glucose syrup comprising the following steps: (a) providing apolypeptide having an amylase and/or glucoamylase activity, wherein thepolypeptide comprises an enzyme of the invention; (b) providing acomposition comprising a polysaccharide, oligosaccharide or starch; and(c) contacting the polypeptide of step (a) and the fabric of step (b)under conditions wherein the polypeptide of step (a) can liquefy thecomposition of step (b) thereby producing a soluble polysaccharide,oligosaccharide or starch, hydrolysate and saccharify the solublepolysaccharide, oligosaccharide or starch, hydrolysate thereby producingthe syrup. In one aspect, the starch can be from rice, corn, barley,wheat, legumes, potato, or sweet potato.

The invention provides methods for improving the flow of thepolysaccharide-comprising, e.g., a starch-containing, production fluidscomprising the following steps: (a) providing a polypeptide having anamylase and/or glucoamylase activity, wherein the polypeptide comprisesa polypeptide of the invention; (b) providing production fluid; and (c)contacting the polypeptide of step (a) and the production fluid of step(b) under conditions wherein the amylase or glucoamylase can hydrolyzethe polysaccharide, oligosaccharide or starch, in the production fluidthereby improving its flow by decreasing its density. In one aspect, theproduction fluid can be from a subterranean formation.

The invention provides anti-staling compositions comprising apolypeptide of the invention or a polypeptide encoded by a nucleic acidof the invention. The invention provides methods for preventing stalingof the baked products comprising the following steps: (a) providing apolypeptide having an amylase and/or glucoamylase activity, wherein thepolypeptide comprises a polypeptide of the invention; (b) providing acomposition containing a polysaccharide, oligosaccharide or starch, usedfor baking; (c) combining the polypeptide of step (a) with thecomposition of the step (b) under conditions wherein the polypeptide canhydrolyze the polysaccharide, oligosaccharide or starch, in thecomposition used for baking thereby preventing staling of the bakedproduct. In one aspect, the baked product can be bread.

The invention provides methods for using amylase or glucoamylase inbrewing or alcohol production comprising the following steps: (a)providing a polypeptide having an amylase and/or glucoamylase activity,wherein the polypeptide comprises a polypeptide of the invention; (b)providing a composition containing a polysaccharide, oligosaccharide orstarch, and used for brewing or in alcohol production; (c) combining thepolypeptide of step (a) with the composition of the step (b) underconditions wherein the polypeptide can hydrolyze the polysaccharide,oligosaccharide or starch, in the composition used for brewing or inalcohol production. In one aspect, the composition containing apolysaccharide, oligosaccharide or starch, can be beer.

The invention provides methods of making a transgenic plant comprisingthe following steps: (a) introducing a heterologous nucleic acidsequence into the cell, wherein the heterologous nucleic sequencecomprises a nucleic acid sequence of the invention, thereby producing atransformed plant cell; and (b) producing a transgenic plant from thetransformed cell. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising the following steps: (a)transforming the plant cell with a heterologous nucleic acid sequenceoperably linked to a promoter, wherein the heterologous nucleic sequencecomprises a nucleic acid of the invention; (b) growing the plant underconditions wherein the heterologous nucleic acids sequence is expressedin the plant cell.

The invention also provides a process for preparing a dough or a bakedproduct prepared from the dough which comprises adding an amylase orglucoamylase of the invention to the dough in an amount which iseffective to retard the staling of the bread. The invention alsoprovides a dough comprising said amylase or glucoamylase and a premixcomprising flour together with said amylase or glucoamylase. Finally,the invention provides an enzymatic baking additive, which contains saidamylase or glucoamylase. The use of the amylase or glucoamylase inaccordance with the present invention provides an improved anti-stalingeffect as measured by, e.g. less crumb firming, retained crumbelasticity, improved slice-ability (e.g. fewer crumbs, non-gummy crumb),improved palatability or flavor.

The enzyme-comprising compositions of the invention (e.g., comprisingpolypeptides, nucleic acids and/or antibodies of this invention) can beformulated in a variety of forms, e.g., as liquids, gels, pills,tablets, sprays, powders, food, feed pellets or encapsulated forms,including nanoencapsulated forms. Any of these embodiments can bedesigned or further formulated as delayed release or “controlledrelease” compositions.

The invention provides delayed release (“controlled release”)compositions comprising an desired ingredient coated by a latex polymer(or equivalent) coating. In one aspect, the desired ingredient comprisesan enzyme, e.g., an enzyme of the invention. In one aspect, a coatedcomposition comprises a drug or pharmaceutical. In one aspect, thedesired ingredient comprises a small molecule, a drug, a polysaccharide,a lipid, a nucleic acid, a vitamin, an antibiotics or an insecticide. Inone aspect, the desired ingredient comprises a pellet or a matrix, e.g.,a pellet or a matrix comprising an edible material (e.g., as an animalfood or feed or supplement or medicament). The invention also providesmethods for the “controlled release” or “delayed release” of acomposition, wherein the composition is coated by a latex polymer (orequivalent) coating.

In one aspect, the latex polymer coating comprises a latex paint, orequivalent. The latex polymer coating can comprise a (meth)acrylate, avinyl acetate, a styrene, an ethylene, a vinyl chloride, a butadiene, avinylidene chloride, a vinyl versatate, a vinyl propionate, a t-butylacrylate, an acrylonitrile, a neoprene, a maleate, a fumarate,equivalents thereof, combinations thereof and/or derivatives thereof.

The invention provides methods for the delayed release or controlledrelease of a composition comprising: (i) (a) providing a composition,and, providing a latex polymer coating; and (b) coating the compositionwith the latex polymer coating; (ii) the method of (i), wherein thecomposition comprises a drug or pharmaceutical; or (iii) the method of(i) or (ii), wherein the composition comprises or consists of thepolypeptide of the invention.

The invention provides oil well drilling fluids comprising thepolypeptides of the invention. The invention provides methods forchanging the viscosity of a composition comprising: (i) (a) providing acomposition and a polypeptide of the invention, and, providing acomposition; and (b) treating the composition with a polypeptide of theinvention; or (ii) the method of (i), wherein the composition comprisesa soil or a drilling mud.

The invention provides methods for aiding in the carrying away ofdrilling mud comprising: (a) providing a composition and a polypeptideof the invention, and a drilling mud; and (b) treating the drilling mudwith a composition comprising a polypeptide of the invention.

The invention provides bio-bleaching solutions comprising a polypeptideof the invention. The invention provides methods for bio-bleaching acomposition comprising: (i) (a) providing a composition and apolypeptide of the invention; and (b) treating the composition with apolypeptide of the invention; or (ii) the method of (i), wherein thecomposition is a paper or a pulp product.

The invention provides methods for making a fuel comprising: (i) (a)providing a polypeptide of the invention; (b) providing a compositioncomprising a polysaccharide, oligosaccharide or starch; and (c)contacting the polypeptide of (a) with the composition of (b) underconditions wherein the polypeptide hydrolyzes the polysaccharide,oligosaccharide or starch; or (ii) the method of (i) wherein thepolypeptide is a thermostable enzyme; (iii) the method of (i) whereinthe fuel is ethanol-based.

The invention provides disinfectants comprising a polypeptide of theinvention.

The invention provides biodefense or bio-detoxifying agents comprising apolypeptide of the invention.

The invention provides dairy products comprising a polypeptide of theinvention.

The invention provides methods for processing a biomass materialcomprising lignocellulose comprising (i) (a) providing a compositioncomprising a polypeptide of the invention, and, providing a biomassmaterial; and (b) contacting the composition comprising a polypeptide ofthe invention, with the biomass material; (ii) the method of (i),wherein the biomass material comprises or is derived from anagricultural crop, or is a byproduct of a food or a feed production, oris a lignocellulosic waste product, or is a plant residue or a wastepaper or waste paper product; (iii) the method of (i) or (ii), whereinthe polypeptide has activity comprising an amylase, a glucoamylase,glucosidase, e.g. alpha-glucosidase or beta-glucosidase activity; (iv)the method of any of (ii), wherein the plant residue comprise stems,leaves, hulls, husks, cobs, wood, wood chips, wood pulp and sawdust, or,the paper waste comprises discarded or used photocopy paper, computerprinter paper, notebook paper, notepad paper, typewriter paper,newspapers, magazines, cardboard and paper-based packaging materials; or(v) the method of (i) to (iv), wherein the processing of the biomassmaterial generates a bioethanol, biopropanol, biobutanol, or abiodiesel; (vi) the method of (i) to (v), wherein the biomass materialcomprises a lignocellulose.

The invention provides biomass materials comprising a polypeptide of theinvention.

The invention provides methods for making bioethanol, biopropanol,biobutanol, or a biodiesel comprising: (i) (a) providing a polypeptideof the invention, and, providing a composition comprising apolysaccharide or oligosaccharide; and (b) contacting the compositioncomprising a polysaccharide or oligosaccharide with a polypeptide of theinvention; (ii) the method of (i), wherein the composition comprising apolysaccharide or oligosaccharide comprises a plant, plant product orplant derivative; (iii) the method of (ii), wherein the plant or plantproduct comprises cane sugar plants or plant products, beets orsugarbeets, wheat, corn, soybeans, potato, rice or barley; (iv) themethod of any of (i) to (iii), wherein the polypeptide has activitycomprising an amylase, a glucoamylase, glucosidase, e.g.alpha-glucosidase or beta-glucosidase activity; or (v) the method of anyof (i) to (iv), wherein the polysaccharide or oligosaccharide comprisesa fermentable sugar.

The invention provides methods for making a fuel comprising (i) (a)providing a polypeptide of the invention, and, providing a compositioncomprising a fermentable sugar; and (b) contacting the compositioncomprising a fermentable sugar with a polypeptide of the invention; (ii)the method of (i), wherein the composition comprising a fermentablesugar comprises a plant, plant product or plant derivative; (iii) themethod of (ii), wherein the plant or plant product comprises cane sugarplants or plant products, beets or sugarbeets, wheat, corn, soybeans,potato, rice or barley; (iv) the method of any of (i) to (iii), whereinthe polypeptide has activity comprising an amylase, a glucoamylase,glucosidase, e.g. alpha-glucosidase or beta-glucosidase activity; or (v)the method of any of (i) to (iv), wherein the fuel comprises abioethanol or a gasoline-ethanol mix, or comprises a bioethanol,biopropanol, biobutanol, or a biodiesel.

The invention provides fuels comprising (a) a polypeptide of theinvention; (b) the fuel of (a), wherein the polypeptide has activitycomprising amylase, glucoamylase, glucosidase, e.g. alpha-glucosidase orbeta-glucosidase activity; (c) the fuel of (a) or (b), wherein the fuelis derived from a plant material, or the fuel is derived from a potato,soybean (rapeseed), barley, rye, corn, oats, wheat, beet or sugar cane;or (d) the fuel of any of (a) to (c), wherein the fuel comprises abioethanol or a gasoline-ethanol mix, or comprises a bioethanol,biopropanol, biobutanol, or a biodiesel.

The invention provides methods for producing a sugar (e.g., amonosaccharide), the method comprising: (i) (a) providing at least onepolypeptide having an amylase or a glucoamylase activity; (b) providinga composition comprising a polysaccharide or an oligosaccharide; and (c)contacting the composition of step (b) with the polypeptide of step (a),thereby generating sugars; (ii) the method of (i), wherein thecomposition comprising a polysaccharide or an oligosaccharide comprisesa starch; (iii) the method of any of (i) or (ii), wherein thepolysaccharide, oligosaccharide and/or sugar comprises or is afermentable sugar; (iv) the method of any of (i), (ii), or (iii),further comprising fermenting the sugar to produce an alcohol; or (v)the method of (iv), wherein the alcohol is an ethanol, propanol orbutanol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 illustrates an exemplary method of the invention for liquefactionsaccharification, as discussed in detail, below.

FIG. 6 illustrates an exemplary corn wet milling process of theinvention using at least one enzyme of the invention, as discussed indetail, below.

FIG. 7, illustrates starch liquefaction.

FIG. 8, illustrates starch liquefaction.

FIG. 9, illustrates starch processing FIG. 10 illustrates the influenceof pH.

FIG. 11 illustrate the influence of pH.

FIG. 12 illustrates how enzymes of the invention can be used in ethanolproduction from corn by dry milling, including their use in both“conventional processes” and “simultaneous liquefaction saccharificationand fermentation processes”, as discussed in detail, below.

FIG. 13, FIG. 14, FIG. 15, FIG. 16, FIG. 17, FIG. 18, FIG. 19 and FIG.20, illustrate rate comparisons of exemplary enzymes of the invention indifferent storage buffers, as discussed in detail in Example 1, below.

FIG. 21 illustrates an SDS PAGE showing the results of proteolysis ofexemplary enzyme SEQ ID NO:52 by pepsin; and

FIG. 22 illustrates the characterization of the peptides generated inthis pepsin digest of SEQ ID NO:52; and

FIG. 23A illustrates the small peptide isolation scheme used for thepeptides generated by the proteolysis of SEQ ID NO:52 by pepsin;

FIG. 23B illustrates an SDS PAGE of the results of the small peptideisolation scheme;

FIG. 23C illustrates the LC/MS profile of the C18 RP eluted fraction;

FIG. 23D illustrates the sequence of the peptides identified by the LCMS/MS analysis;

FIG. 23E and FIG. 23F illustrate the “Asn-Xaa-Ser/Thr” sequins (motifs)in the sequence output (highlighted in blue); asparagines predicted tobe N-glycosylated are highlighted in red, as discussed in detail inExample 22, below.

FIG. 24 illustrates Table 1, which summarizes data comparing initialrates of granular corn starch and soluble starch (dextrin) hydrolysis byexemplary enzymes of the invention and a benchmark enzyme A. nigerglucoamylase, as discussed in detail in Example 23, below.

FIG. 25 illustrates the effect of temperature on the activity of theexemplary glucoamylase SEQ ID NO:20 and the A. niger “benchmark” enzymewith soluble starch (dextrin) as a substrate, as discussed in detail inExample 23, below.

FIG. 26 illustrates tables summarizing the efficiency of exemplaryenzyme purification protocols on selected enzymes of this invention, andcorresponding activity data on, inter alia, raw starch and solublestarch comparing purified and unpurified enzyme, as discussed in detailin Example 18, below.

FIG. 27 illustrates the theoretical basis of the CERALPHA™ alpha-amylaseassay procedure, as discussed in detail in Example 27, below.

FIG. 28A shows data demonstrating the effect of temperature on theactivity of e.g., an exemplary glucoamylase of the invention withgranular starch as a substrate, as discussed in detail in Example 29,below.

FIG. 28B shows data demonstrating the effect of temperature on theactivity of e.g., an exemplary glucoamylase of the invention withsoluble starch (dextrin) as a substrate, as discussed in detail inExample 29, below.

FIG. 29A shows data demonstrating the effect of pH on the activity ofe.g., an exemplary glucoamylase of the invention with granular starch asa substrate, as discussed in detail in Example 29, below.

FIG. 29B shows data demonstrating the effect of pH on the activity ofe.g., an exemplary glucoamylase of the invention with soluble starch(dextrin) as a substrate, as discussed in detail in Example 29, below.

FIG. 30 shows data demonstrating the effect of temperature on starchhydrolysis by the characterized α-amylases, as discussed in detail inExample 29, below.

FIG. 31A shows data demonstrating the effect pH on the activities ofexemplary alpha-Amylases and/or glucoamylases of the invention withGranular Starch as substrate, as discussed in detail in Example 29,below.

FIG. 31B shows data demonstrating the effect pH on the activities ofexemplary alpha-Amylases and/or glucoamylases of the invention withSoluble Starch as substrate, as discussed in detail in Example 29,below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION

The invention provides amylase enzymes, e.g., an alpha amylases,polynucleotides encoding the enzymes, methods of making and using thesepolynucleotides and polypeptides. The invention is directed to novelpolypeptides having an amylase and/or glucoamylase activity, e.g., analpha amylase activity, nucleic acids encoding them and antibodies thatbind to them. The polypeptides of the invention can be used in a varietyof diagnostic, therapeutic, and industrial contexts. The polypeptides ofthe invention can be used as, e.g., an additive for a detergent, forprocessing foods and for chemical synthesis utilizing a reversereaction. Additionally, the polypeptides of the invention can be used infabric treatment, alcohol production, and as additives to food or animalfeed.

In one aspect, the amylases and/or glucoamylases of the invention areactive at a high and/or at a low temperature, or, over a wide range oftemperature. For example, they can be active in the temperatures rangingbetween 20° C. to 90° C., between 30° C. to 80° C., or between 40° C. to70° C. The invention also provides amylases that have activity atalkaline pHs or at acidic pHs, e.g., low water acidity. In alternativeaspects, the Amylases and/or glucoamylases of the invention can haveactivity in acidic pHs as low as pH 5.5, pH 5.0, pH 4.5, pH 4.0, and pH3.5. In alternative aspects, the Amylases and/or glucoamylases of theinvention can have activity in alkaline pHs as high as pH 8, pH 9.5, pH10, pH 10.5, and pH 11. In one aspect, the Amylases and/or glucoamylasesof the invention are active in the temperature range of between about40° C. to about 70° C. under conditions of low water activity (low watercontent). For example, the invention provides amylases, includingglucoamylases, with the ability to hydrolyze a polysaccharide,oligosaccharide or starch, e.g., a granular starch (including rawgranular starch), at low temperatures, e.g., in the range of about 30°C. to 40° C.; at low pH, e.g., in the range of about pH 3.5 to pH 6.0;and, at low temperatures and low pH, e.g., in the range of about 30° C.to 40° C. and at low pH in the range of about pH 3.5 to pH 6.0.

The invention also provides methods for further modifying the exemplaryAmylases and/or glucoamylases of the invention to generate proteins withdesirable properties. For example, amylases generated by the methods ofthe invention can have altered enzymatic activity, thermal stability,pH/activity profile, pH/stability profile (such as increased stabilityat low, e.g. pH<6 or pH<5, or high, e.g. pH>9, pH values), stabilitytowards oxidation, Ca′ dependency, specific activity and the like. Theinvention provides for altering any property of interest. For instance,the alteration may result in a variant which, as compared to a parentenzyme, has altered enzymatic activity, or, pH or temperature activityprofiles.

Generating and Manipulating Nucleic Acids

In one aspect, the invention provides isolated, synthetic or recombinantnucleic acids comprising a nucleic acid sequence having at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%)sequence identity to an exemplary nucleic acid of the invention over aregion of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550 or more, residues. In one aspect, the nucleicacid encodes at least one polypeptide having an amylase and/orglucoamylase activity, e.g., an alpha amylase activity.

“Synthetic” nucleic acids (including oligonucleotides), polypeptides orproteins of the invention include those prepared by any chemicalsynthesis, e.g., as described, below.

The phrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotides, nucleotides, polynucleotides, or to a fragment of anyof these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic, recombinantor synthetic origin which may be single-stranded or double-stranded andmay represent a sense or antisense strand, to peptide nucleic acid(PNA), or to any DNA-like or RNA-like material, natural or synthetic inorigin, including, e.g., iRNA such as miRNA or siRNA, ribonucleoproteins(e.g., iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156.

The invention provides “recombinant” and synthetic nucleic acids, whichcan include nucleic acids adjacent to a “backbone” nucleic acid to whichit is not adjacent in its natural environment. In one aspect, nucleicacids represent 5% or more of the number of nucleic acid inserts in apopulation of nucleic acid “backbone molecules.” “Backbone molecules”according to the invention include nucleic acids such as expressionvectors, self-replicating nucleic acids, viruses, integrating nucleicacids, and other vectors or nucleic acids used to maintain or manipulatea nucleic acid insert of interest. In one aspect, the enriched nucleicacids represent 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules. “Recombinant” polypeptides or proteins refer to polypeptidesor proteins produced by recombinant DNA techniques; e.g., produced fromcells transformed by an exogenous DNA construct encoding the desiredpolypeptide or protein.

“Oligonucleotide” includes either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands, which may bechemically synthesized (i.e., as synthetic nucleic acids). Inalternative embodiments, synthetic nucleic acids and oligonucleotides ofthe invention have no 5′ phosphate and thus will not ligate to anotheroligonucleotide without adding a phosphate with an ATP in the presenceof a kinase. In alternative embodiments, a synthetic oligonucleotide canligate to a fragment that has not been dephosphorylated.

The term “gene” includes a nucleic acid sequence comprising a segment ofDNA involved in producing a transcription product (e.g., a message),which in turn is translated to produce a polypeptide chain, or regulatesgene transcription, reproduction or stability. Genes can include regionspreceding and following the coding region, such as leader and trailer,promoters and enhancers, as well as, where applicable, interveningsequences (introns) between individual coding segments (exons).

The invention provides isolated and recombinant nucleic acids, includingexpression cassettes such as expression vectors encoding thepolypeptides of the invention. The invention provides probes comprisingor consisting of nucleic acids of the invention. The invention alsoincludes methods for discovering new amylase sequences using the nucleicacids of the invention. The invention also includes methods forinhibiting the expression of amylase genes, transcripts and polypeptidesusing the nucleic acids of the invention. Also provided are methods formodifying the nucleic acids of the invention by, e.g., syntheticligation reassembly, optimized directed evolution system and/or genesite saturation mutagenesis (GSSM).

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like. Inpracticing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides generated from these nucleic acids can beindividually isolated or cloned and tested for a desired activity. Anyrecombinant expression system can be used, including bacterial,mammalian, yeast, insect or plant cell expression systems.

The invention provides methods for optimizing amylase enzymes andenzyme-encoding nucleic acid sequences, e.g., making “variantsequences”, comprising use of sequences of the invention using, e.g.,“saturation mutagenesis” or “GSSM” (includes a method that usesdegenerate oligonucleotide primers to introduce point mutations into apolynucleotide, as described in detail, below); “optimized directedevolution system” or “optimized directed evolution” (includes a methodfor reassembling fragments of related nucleic acid sequences, e.g.,related genes, and explained in detail, below) and/or “syntheticligation reassembly” or “SLR” (includes a method of ligatingoligonucleotide fragments in a non-stochastic fashion, and explained indetail, below). “Variant” includes polynucleotides or polypeptides ofthe invention modified at one or more base pairs, codons, introns,exons, or amino acid residues (respectively) yet still retain thebiological activity of an amylase and/or glucoamylase of the invention.Variants can be produced by any number of means included methods suchas, for example, error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, GSSMand any combination thereof. Techniques for producing variant amylasehaving activity at a pH or temperature, for example, that is differentfrom an exemplary enzyme of this invention, or a wild-type amylase, areprovided herein.

Nucleic acids of the invention can be completely or partially synthetic,and in alternative aspects, they can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. Promoters that are used topractice this invention include all sequences capable of drivingtranscription of a coding sequence in a cell, e.g., a plant cell. Thus,promoters used in the constructs of the invention include cis-actingtranscriptional control elements and regulatory sequences that areinvolved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter used to practice thisinvention can be a cis-acting transcriptional control element, includingan enhancer, a promoter, a transcription terminator, an origin ofreplication, a chromosomal integration sequence, 5′ and 3′ untranslatedregions, or an intronic sequence, which are involved in transcriptionalregulation. These cis-acting sequences typically interact with proteinsor other biomolecules to carry out (turn on/off, regulate, modulate,etc.) transcription. “Constitutive” promoters used to practice thisinvention can be those that drive expression continuously under mostenvironmental conditions and states of development or celldifferentiation. Promoters used to practice this invention can be“inducible” or “regulatable” promoters direct expression of the nucleicacid of the invention under the influence of environmental conditions ordevelopmental conditions. Examples of environmental conditions that mayaffect transcription by inducible promoters include anaerobicconditions, elevated temperature, drought, or the presence of light.

In one embodiment, a promoter sequence can be “operably linked to” acoding sequence of the invention, e.g., when RNA polymerase whichinitiates transcription at the promoter will transcribe the codingsequence into mRNA.

Promoters used to practice this invention include “tissue-specific”promoters, which are transcriptional control elements that are onlyactive in particular cells or tissues or organs, e.g., in plants oranimals. Tissue-specific regulation may be achieved by certain intrinsicfactors which ensure that genes encoding proteins specific to a giventissue are expressed. Such factors are known to exist in mammals andplants so as to allow for specific tissues to develop.

The expression control sequence(s) used to practice this invention canbe in an expression vector. Exemplary bacterial promoters include lacI,lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplary eukaryotic promotersinclude CMV immediate early, HSV thymidine kinase, early and late SV40,LTRs from retrovirus, and mouse metallothionein I. Promoters suitablefor expressing a polypeptide in bacteria include the E. coli lac or trppromoters, the lacI promoter, the lacZ promoter, the T3 promoter, the T7promoter, the gpt promoter, the lambda PR promoter, the lambda PLpromoter, promoters from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), and the acid phosphatase promoter.Eukaryotic promoters include the CMV immediate early promoter, the HSVthymidine kinase promoter, heat shock promoters, the early and late SV40promoter, LTRs from retroviruses, and the mouse metallothionein-Ipromoter. Other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses may also be used.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express an amylase and/orglucoamylase of the invention in a tissue-specific manner. The inventionalso provides plants or seeds that express an amylase and/or aglucoamylase of the invention in a tissue-specific manner. Thetissue-specificity can be seed specific, stem specific, leaf specific,root specific, fruit specific and the like.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassava vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression ofamylase-expressing nucleic acid in a specific tissue, organ or cell type(i.e. tissue-specific promoters) or may be otherwise under more preciseenvironmental or developmental control or under the control of aninducible promoter. Examples of environmental conditions that may affecttranscription include anaerobic conditions, elevated temperature, thepresence of light, or sprayed with chemicals/hormones. For example, theinvention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fbl2A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically—(e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato, sugar cane, sugar beet, or other crops, inducibleat any stage of development of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the amylase-producing nucleic acids of the invention willallow the grower to select plants with the optimal starch/sugar ratio.The development of plant parts can thus controlled. In this way theinvention provides the means to facilitate the harvesting of plants andplant parts. For example, in various embodiments, the maize In2-2promoter, activated by benzenesulfonamide herbicide safeners, is used(De Veylder (1997) Plant Cell Physiol. 38:568-577); application ofdifferent herbicide safeners induces distinct gene expression patterns,including expression in the root, hydathodes, and the shoot apicalmeristem. Coding sequences of the invention are also under the controlof a tetracycline-inducible promoter, e.g., as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324).

If proper polypeptide expression is desired, a polyadenylation region atthe 3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from genes in the Agrobacterial T-DNA.

The invention provides “expression cassettes” comprising any sequence ofthe invention “operably linked” to a transcriptional regulator; e.g.,wherein “operably linked” as used herein refers to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments.Typically, it refers to the functional relationship of transcriptionalregulatory sequence to a transcribed sequence. For example, a promoteris operably linked to a coding sequence, such as a nucleic acid of theinvention, if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

Modification of Coding Sequences and Adjacent Sequences

The transgenic expression in plants of genes derived from heterologoussources may involve the modification of those genes to achieve andoptimize their expression in plants. In particular, bacterial ORFs whichencode separate enzymes but which are encoded by the same transcript inthe native microbe are best expressed in plants on separate transcripts.To achieve this, each microbial ORF is isolated individually and clonedwithin a cassette which provides a plant promoter sequence at the 5′ endof the ORF and a plant transcriptional terminator at the 3′ end of theORF. The isolated ORF sequence preferably includes the initiating ATGcodon and the terminating STOP codon but may include additional sequencebeyond the initiating ATG and the STOP codon. In addition, the ORF maybe truncated, but still retain the required activity; for particularlylong ORFs, truncated versions which retain activity may be preferablefor expression in transgenic organisms. By “plant promoter” and “planttranscriptional terminator” it is intended to mean promoters andtranscriptional terminators which operate within plant cells. Thisincludes promoters and transcription terminators which may be derivedfrom non-plant sources such as viruses (an example is the CauliflowerMosaic Virus).

In some cases, modification to the ORF coding sequences and adjacentsequence is not required. It is sufficient to isolate a fragmentcontaining the ORF of interest and to insert it downstream of a plantpromoter. For example, Gaffney et. al. (Science 261: 754-756 (1993))have expressed the Pseudomonas nahG gene in transgenic plants under thecontrol of the CaMV 35S promoter and the CaMV tml terminatorsuccessfully without modification of the coding sequence and withnucleotides of the Pseudomonas gene upstream of the ATG still attached,and nucleotides downstream of the STOP codon still attached to the nahGORF. Preferably as little adjacent microbial sequence should be leftattached upstream of the ATG and downstream of the STOP codon. Inpractice, such construction may depend on the availability ofrestriction sites.

In other cases, the expression of genes derived from microbial sourcesmay provide problems in expression. These problems have been wellcharacterized in the art and are particularly common with genes derivedfrom certain sources, such as Bacillus. These problems may apply to thenucleotide sequence of this invention and the modification of thesegenes can be undertaken using techniques now well known in the art. Thefollowing problems may be encountered:

Codon Usage

The preferred codon usage in plants differs from the preferred codonusage in certain microorganisms. Comparison of the usage of codonswithin a cloned microbial ORF to usage in plant genes (and in particulargenes from the target plant) will enable an identification of the codonswithin the ORF which should preferably be changed. Typically plantevolution has tended towards a strong preference of the nucleotides Cand G in the third base position of monocotyledons, whereas dicotyledonsoften use the nucleotides A or T at this position. By modifying a geneto incorporate preferred codon usage for a particular target transgenicspecies, many of the problems described below for GC/AT content andillegitimate splicing will be overcome.

GC/AT Content

Plant genes typically have a GC content of more than 35%. ORF sequenceswhich are rich in A and T nucleotides can cause several problems inplants. Firstly, motifs of ATTTA are believed to cause destabilizationof messages and are found at the 3′ end of many short-lived mRNAs.Secondly, the occurrence of polyadenylation signals such as AATAAA atinappropriate positions within the message is believed to causepremature truncation of transcription. In addition, monocotyledons mayrecognize AT-rich sequences as splice sites (see below).

Sequences Adjacent to the Initiating Methionine

Plants differ from microorganisms in that their messages do not possessa defined ribosome binding site. Rather, it is believed that ribosomesattach to the 5′ end of the message and scan for the first available ATGat which to start translation. Nevertheless, it is believed that thereis a preference for certain nucleotides adjacent to the ATG and thatexpression of microbial genes can be enhanced by the inclusion of aeukaryotic consensus translation initiator at the ATG. Clontech(1993/1994 catalog, page 210) have suggested one sequence as a consensustranslation initiator for the expression of the E. coli uidA gene inplants. Further, Joshi, N.A.R. 15: 6643-6653 (1987), has compared manyplant sequences adjacent to the ATG and suggests another consensussequence. In situations where difficulties are encountered in theexpression of microbial ORFs in plants, inclusion of one of thesesequences at the initiating ATG may improve translation. In such casesthe last three nucleotides of the consensus may not be appropriate forinclusion in the modified sequence due to their modification of thesecond AA residue. Preferred sequences adjacent to the initiatingmethionine may differ between different plant species. A survey of 14maize genes located in the GenBank database provided the followingresults:

Position Before the Initiating ATG in 14 Maize Genes: −10 −9 −8 −7 −6 −5−4 −3 −2 −1 C3 8 4 6 2 5 6 0 10 7 T3 0 3 4 3 2 1 1 1 0 A2 3 1 4 3 2 3 72 3 G6 3 6 0 6 5 4 6 1 5

This analysis can be done for the desired plant species into which thenucleotide sequence is being incorporated, and the sequence adjacent tothe ATG modified to incorporate the preferred nucleotides.

Removal of Illegitimate Splice Sites

Genes cloned from non-plant sources and not optimized for expression inplants may also contain motifs which may be recognized in plants as 5′or 3′ splice sites, and be cleaved, thus generating truncated or deletedmessages. These sites can be removed using the techniques well known inthe art.

Techniques for the modification of coding sequences and adjacentsequences are well known in the art. In cases where the initialexpression of a microbial ORF is low and it is deemed appropriate tomake alterations to the sequence as described above, then theconstruction of synthetic genes can be accomplished according to methodswell known in the art. These are, for example, described in thepublished patent disclosures EP 0 385 962 (to Monsanto), EP 0 359 472(to Lubrizol) and WO 93/07278 (to Ciba-Geigy). In most cases it ispreferable to assay the expression of gene constructions using transientassay protocols (which are well known in the art) prior to theirtransfer to transgenic plants.

Plant Promoters

The compositions of the invention may contain nucleic acid sequences fortransformation and expression in a plant of interest. The nucleic acidsequences may be present in DNA constructs or expression cassettes. Inalternative embodiments, “expression cassette” as used herein means anucleic acid molecule capable of directing expression of a particularnucleotide sequence in an appropriate host cell, comprising a promoteroperatively linked to the nucleotide sequence of interest, which isoperatively linked to termination signals. It also typically comprisessequences required for proper translation of the nucleotide sequence.The coding region usually codes for a protein of interest but may alsocode for a functional RNA of interest, for example antisense RNA or anontranslated RNA, in the sense or antisense direction. The expressioncassette comprising the nucleotide sequence of interest may be chimeric,meaning that at least one of its components is heterologous with respectto at least one of its other components. The expression cassette mayalso be one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression. In alternativeembodiments, the expression cassette is heterologous with respect to thehost, i.e., the particular DNA sequence of the expression cassette doesnot occur naturally in the host cell and must have been introduced intothe host cell or an ancestor of the host cell by a transformation event.The expression of the nucleotide sequence in the expression cassette maybe under the control of a constitutive promoter or of an induciblepromoter that initiates transcription only when the host cell is exposedto some particular external stimulus. In alternative embodiments, thepromoter can also be specific to a particular tissue or organ or stageof development.

The present invention encompasses the transformation of plants withexpression cassettes capable of expressing polynucleotides. Inalternative embodiments, the expression cassette will include in the5′-3′ direction of transcription, a transcriptional and translationalinitiation region (i.e., a promoter) and a polynucleotide of interest.The expression cassette may optionally comprise a transcriptional andtranslational termination region (i.e. termination region) functional inplants. In some embodiments, the expression cassette comprises aselectable marker gene to allow for selection for stable transformants.Expression constructs of the invention may also comprise a leadersequence and/or a sequence allowing for inducible expression of thepolynucleotide of interest. See, Guo et. al. (2003) Plant J. 34:383-92and Chen et. al. (2003) Plant J. 36:731-40 for examples of sequencesallowing for inducible expression.

In alternative embodiments, the regulatory sequences of the expressionconstruct are operably linked to the polynucleotide of interest. By“operably linked” is intended a functional linkage between a promoterand a second sequence wherein the promoter sequence initiates andmediates transcription of the DNA sequence corresponding to the secondsequence. In alternative embodiments, operably linked means that thenucleotide sequences being linked are contiguous.

Any promoter capable of driving expression in the plant of interest maybe used in the practice of the invention. The promoter may be native oranalogous or foreign or heterologous to the plant host. In alternativeembodiments, the terms “heterologous” and “exogenous” when used hereinto refer to a nucleic acid sequence (e.g. a DNA or RNA sequence) or agene, refer to a sequence that originates from a source foreign to theparticular host cell or, if from the same source, is modified from itsoriginal form. Thus, a heterologous gene in a host cell includes a genethat is endogenous to the particular host cell but has been modified.The terms also include non-naturally occurring multiple copies of anaturally occurring DNA sequence. Thus, the terms refer to a DNA segmentthat is foreign or heterologous to the cell, or homologous to the cellbut in a position within the host cell nucleic acid in which the elementis not ordinarily found. Exogenous DNA segments are expressed to yieldexogenous polypeptides.

In alternative embodiments, a “homologous” nucleic acid (e.g. DNA)sequence is a nucleic acid (e.g. DNA or RNA) sequence naturallyassociated with a host cell into which it is introduced.

The choice of promoters to be included can depend upon several factors,including, but not limited to, efficiency, selectability, inducibility,desired expression level, and cell- or tissue-preferential expression.It is a routine matter for one of skill in the art to modulate theexpression of a sequence by appropriately selecting and positioningpromoters and other regulatory regions relative to that sequence.

In alternative embodiments, suitable promoters initiate transcriptiononly, or predominantly, in certain cell types. Thus, as used herein acell type- or tissue-preferential promoter is one that drives expressionpreferentially in the target tissue, but may also lead to someexpression in other cell types or tissues as well. Methods foridentifying and characterizing promoter regions in plant genomic DNAinclude, for example, those described in the following references:Jordano, et. al., Plant Cell, 1:855-866 (1989); Bustos, et. al., PlantCell, 1:839-854 (1989); Green, et. al., EMBO J. 7, 4035-4044 (1988);Meier, et. al., Plant Cell, 3, 309-316 (1991); and Zhang, et. al., PlantPhysiology 110: 1069-1079 (1996).

In alternative embodiments, tissue preferred regulated genes and/orpromoters which have been reported in plants can be used. Reportedtissue preferred genes that can be used in alternative embodimentsinclude the genes encoding the seed storage proteins (such as napin,cruciferin, beta-conglycinin, and phaseolin, prolamines, glutelins,globulins, and zeins) zeins or oil body proteins (such as oleosin), orgenes involved in fatty acid biosynthesis (including acyl carrierprotein, stearoyl-ACP desaturase, and fatty acid desaturases (fad 2-1)),and other genes expressed during embryo development (such as Bce4, see,for example, EP 255378 and Kridl et. al., (1991) Seed Science Research,1:209). Examples of tissue-specific promoters that can be used topractice this invention, which have been described, include the lectin(Vodkin, Prog. Clin. Biol. Res., 138; 87 (1983); Lindstrom et. al.,(1990) Der. Genet., 11:160), corn alcohol dehydrogenase 1 (Dennis et.al., Nucleic Acids Res., 12:3983 (1984)), corn light harvesting complex(see, e.g., Simpson, (1986) Science, 233:34; Bansal (1992) Proc. Natl.Acad. Sci. USA 89:3654), corn heat shock protein (see, e.g., Odell et.al., (1985) Nature, 313:810; pea small subunit RuBP carboxylase (see,e.g., Poulsen et. al., (1986) Mol. Gen. Genet., 205:193-200; Cashmoreet. al., (1983) Gen. Eng. of Plants, Plenum Press, New York, 29-38); Tiplasmid mannopine synthase (see, e.g., Langridge et. al., (1989) Proc.Natl. Acad. Sci. USA, 86:3219-3223), Ti plasmid nopaline synthase(Langridge et. al., (1989) Proc. Natl. Acad. Sci. USA, 86:3219-3223),petunia chalcone isomerase (see, e.g., vanTunen (1988) EMBO J. 7:1257);bean glycine rich protein 1 (see, e.g., Keller (1989) Genes Dev.3:1639); truncated CaMV 35s (see, e.g., Odell (1985) Nature 313:810);potato patatin (see, e.g., Wenzler (1989) Plant Mol. Biol. 13:347; rootcell (see, e.g., Yamamoto (1990) Nucleic Acids Res. 18:7449); maize zein(see, e.g., Reina (1990) Nucleic Acids Res. 18:6425; Lopes et. al.(1995) Mol. Gen. Genet. 247: 603-613; Kriz (1987) Mol. Gen. Genet.207:90; Wandelt (1989) Nucleic Acids Res., 17:2354; Langridge (1983)Cell, 34:1015; Reina (1990) Nucleic Acids Res., 18:7449), ADP-gpppromoter (see, e.g., U.S. Pat. No. 7,102,057); globulin-1 (see, e.g.,Belanger (1991) Genetics 129:863); alpha-globulin (Sunilkumar, et. al.(2002), Transgenic Res. 11:347-359); α-tubulin; cab (see, e.g., Sullivan(1989) Mol. Gen. Genet., 215:431); PEPCase (see e.g., Hudspeth & Grula,(1989) Plant Molec. Biol., 12:579-589); R gene complex-associatedpromoters (Chandler et. al., (1989) Plant Cell, 1:1175); pea vicilinpromoter (Czako et. al., (1992) Mol. Gen. Genet., 235:33; U.S. Pat. No.5,625,136); GTL1 promoter (Takaiwa et. al. (1991) Plant Mol. Biol. 16(1), 49-58); chalcone synthase promoters (Franken et. al., (1991) EMBOJ., 10:2605); and/or a GY1 promoter (Sims & Goldburg (1989) Nuc. AcidRes. 17(11) 4368) and the like.

In alternative embodiments, a class of fruit-preferred promotersexpressed at or during antithesis through fruit development, at leastuntil the beginning of ripening, is used, e.g., as discussed in U.S.Pat. No. 4,943,674. The promoter for polygalacturonase gene is active infruit ripening. A polygalacturonase gene also can be used, e.g., asdescribed in U.S. Pat. Nos. 4,535,060, 4,769,061, 4,801,590, and5,107,065.

Other examples of tissue-preferred promoters that can be used includethose that direct expression in leaf cells following damage to the leaf(for example, from chewing insects), in tubers (for example, patatingene promoter), and in fiber cells (an example of adevelopmentally-regulated fiber cell protein is E6 (John & Crow (1992)PNAS 89:5769-5773). The E6 gene is most active in fiber, although lowlevels of transcripts are found in leaf, ovule and flower.

In alternative embodiments, promoters active in photosynthetic tissuecan be used in order to drive transcription in green tissues such asleaves and stems; these promoters are suitable when they driveexpression only or predominantly in such tissues. In alternativeembodiments, the promoter may confer expression constitutivelythroughout the plant, or differentially with respect to the greentissues, or differentially with respect to the developmental stage ofthe green tissue in which expression occurs, or in response to externalstimuli.

Examples of promoters that can be used to practice this inventioninclude the ribulose-1,5-bisphosphate carboxylase (RbcS) promoters suchas the RbcS promoter from eastern larch (Larix laricina), the pine cab6promoter (Yamamoto et. al. (1994) Plant Cell Physiol. 35:773-778), theCab-1 gene promoter from wheat (Fejes et. al. (1990) Plant Mol. Biol.15:921-932), the CAB-1 promoter from spinach (Lubberstedt et. al. (1994)Plant Physiol. 104:997-1006), the cab1R promoter from rice (Luan et. al.(1992) Plant Cell 4:971-981), the pyruvate orthophosphate dikinase(PPDK) promoter from corn (Matsuoka et. al. (1993) Proc. Natl. Acad. SciUSA 90:9586-9590), the tobacco Lhcb1*2 promoter (Cerdan et. al. (1997)Plant Mol. Biol. 33:245-255), the Arabidopsis thaliana SUC2 sucrose-H+symporter promoter (Truernit et. al. (1995) Planta 196:564-570), andthylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC,FNR, atpC, atpD, cab, rbcS. Other promoters that drive transcription instems, leafs and green tissue are described in U.S. Patent PublicationNo. 2007/0006346.

The tissue specificity of some “tissue preferred” promoters may not beabsolute; in alternative embodiments reporter genes such as Gus or greenfluorescent protein, cyan fluorescent protein, yellow fluorescentprotein or red fluorescent protein are used. In alternative embodimentstissue preferred expression can be achieved with “leaky” expression by acombination of different tissue-preferred promoters. Other tissuepreferred promoters can be used, and they can be isolated by one skilledin the art (see U.S. Pat. No. 5,589,379).

In one aspect, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotic. Forexample, gene expression systems that are activated in the presence of achemical ligand, including ethanol, such as can be found in WO 96/27673;WO 93/01294; WO 94/03619; WO 02/061102, all of which are herebyincorporated by reference. The maize In2-2 promoter, activated bybenzenesulfonamide herbicide safeners, can be used (De Veylder (1997)Plant Cell Physiol. 38:568-577); application of different herbicidesafeners induces distinct gene expression patterns, including expressionin the root, hydathodes, and the shoot apical meristem. Coding sequencecan be under the control of, e.g., a tetracycline-inducible promoter,e.g., as described with transgenic tobacco plants containing the Avenasativa L. (oat) arginine decarboxylase gene (Masgrau (1997) Plant J.11:465-473); estrogen, such as, the ecdysone receptor (WO 01/52620) or,a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically—(e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant.

Examples of some constitutive promoters which can be used to practicethis invention, and which have been described, include rice actin 1(Wang et. al. (1992) Mol. Cell. Biol., 12:3399; U.S. Pat. No.5,641,876); other actin isoforms (McElroy et. al. (1990) Plant Cell 2:163-171 and McElroy et. al. (1991) Mol. Gen. Genet. 231: 150-160); CaMV35S (Odell et. al. (1985) Nature, 313:810); CaMV 19S (Lawton et. al.(1987) Plant Mol. Biol. 9:315-324; U.S. Pat. No. 5,639,949); nos (Ebertet. al. (1987) PNAS USA 84:5745-5749); Adh (Walker et. al. (1987) PNASUSA 84:6624-6628), sucrose synthase (Yang & Russell (1990) PNAS USA87:4144-4148); and the ubiquitin promoters (e.g. sunflower—Binet et. al.(1991) Plant Science 79: 87-94; maize—Christensen et. al. (1989) PlantMolec. Biol. 12: 619-632; and Arabidopsis—Callis et. al., J. Biol. Chem.(1990) 265:12486-12493; and Norris et. al., Plant Mol. Biol. (1993)21:895-906.

A variety of transcriptional terminators can be used in expressioncassettes to practice this invention. These transcriptional terminatorsare responsible for the termination of transcription beyond thetransgene and correct mRNA polyadenylation. The termination region maybe native with the transcriptional initiation region, may be native withthe operably linked DNA sequence of interest, may be native with theplant host, or may be derived from another source (i.e., foreign orheterologous to the promoter, the DNA sequence of interest, the planthost, or any combination thereof). Appropriate transcriptionalterminators are those that are known to function in plants and includethe CAMV 35S terminator, the tml terminator, the nopaline synthaseterminator and the pea rbcs E9 terminator. These can be used in bothmonocotyledons and dicotyledons. In addition, a gene's nativetranscription terminator may be used.

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants. For example, various intron sequenceshave been shown to enhance expression, particularly in monocotyledonouscells. For example, the introns of the maize Adh1 gene have been foundto significantly enhance the expression of the wild-type gene under itscognate promoter when introduced into maize cells.

In alternative embodiments, non-translated leader sequences derived fromviruses can be used to enhance expression, and these are particularlyeffective in dicotyledonous cells. In alternative embodiments, leadersequences from Tobacco Mosaic Virus (TMV, the “W-sequence”), MaizeChlorotic Mottle Virus (MCMV), and Alfalfa Mosaic Virus (AMV) are used,and these have been shown to be effective in enhancing expression (e.g.Gallie et. al. Nucl. Acids Res. 15: 8693-8711 (1987); Skuzeski et. al.Plant Molec. Biol. 15: 65-79 (1990)).

Targeting of the Gene Product within the Cell

In alternative embodiments, various mechanisms for targeting geneproducts are used; and these are known to exist in plants, and thesequences controlling the functioning of these mechanisms have beencharacterized in some detail. Sequences have been characterized whichcause the targeting of gene products to other cell compartments. Aminoterminal sequences can be responsible for targeting a protein ofinterest to any cell compartment, such as, a vacuole, mitochondrion,peroxisome, protein bodies, endoplasmic reticulum, chloroplast, starchgranule, amyloplast, apoplast or cell wall of a plant (e.g. Unger et.al. Plant Molec. Biol. 13: 411-418 (1989); Rogers et. al. (1985) Proc.Natl. Acad. Sci. USA 82: 6512-651; U.S. Pat. No. 7,102,057; WO2005/096704, all of which are hereby incorporated by reference). Inalternative embodiments, various signal sequence are used, e.g., thesignal sequence may be an N-terminal signal sequence from waxy, anN-terminal signal sequence from γ-zein, a starch binding domain, aC-terminal starch binding domain, a chloroplast targeting sequence,which imports the mature protein to the chloroplast (Comai et. al.(1988) J. Biol. Chem. 263: 15104-15109; van den Broeck, et. al. (1985)Nature 313: 358-363; U.S. Pat. No. 5,639,949) or a secretion signalsequence from aleurone cells (Koehler & Ho, Plant Cell 2: 769-783(1990)). In alternative embodiments, amino terminal sequences are usedin conjunction with carboxy terminal sequences; these are responsiblefor vacuolar targeting of gene products (Shinshi et. al. (1990) PlantMolec. Biol. 14: 357-368).

The signal sequence selected can include the known cleavage site, andthe fusion constructed should take into account any amino acids afterthe cleavage site(s), which are required for cleavage. In some casesthis requirement may be fulfilled by the addition of a small number ofamino acids between the cleavage site and the transgene ATG or,alternatively, replacement of some amino acids within the transgenesequence. These construction techniques are well known in the art andare equally applicable to any cellular compartment.

The above-described mechanisms for cellular targeting can be utilizednot only in conjunction with their cognate promoters, but also inconjunction with heterologous promoters so as to effect a specificcell-targeting goal under the transcriptional regulation of a promoterthat has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding theamylases and/or glucoamylases of the invention. Expression vectors andcloning vehicles of the invention can comprise viral particles,baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterialartificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul poxvirus, pseudorabies and derivatives of SV40), P1-based artificialchromosomes, yeast plasmids, yeast artificial chromosomes, and any othervectors specific for specific hosts of interest (such as bacillus,Aspergillus and yeast). Vectors of the invention can includechromosomal, non-chromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. Exemplary vectors are include: bacterial: pQEvectors (Qiagen), pBLUESCRIPT™ (pBluescript) plasmids, pNH vectors,(lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T(Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG,pSVLSV40 (Pharmacia). However, any other plasmid or other vector may beused so long as they are replicable and viable in the host. Low copynumber or high copy number vectors may be employed with the presentinvention. “Plasmids” can be commercially available, publicly availableon an unrestricted basis, or can be constructed from available plasmidsin accord with published procedures. Equivalent plasmids to thosedescribed herein are known in the art and will be apparent to theordinarily skilled artisan.

The invention provides “expression cassettes” comprising any sequence ofthe invention “operably linked” to a transcriptional regulator; the term“expression cassette” as used herein can refer to a nucleotide sequencewhich is capable of affecting expression of a structural gene (e.g., aprotein coding sequence, such as an amylase and/or a glucoamylase of theinvention) in a host compatible with such sequences. Expressioncassettes include at least a promoter operably linked with thepolypeptide coding sequence; and, optionally, with other sequences,e.g., transcription termination signals. Additional factors necessary orhelpful in effecting expression may also be used, e.g., enhancers. Thus,expression cassettes also include plasmids, expression vectors,recombinant viruses, any form of recombinant “naked DNA” vector, and thelike.

A “vector” comprises a nucleic acid which can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector optionally comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors used to practice this inventioninclude, but are not limited to replicons (e.g., RNA replicons,bacteriophages) to which fragments of DNA may be attached and becomereplicated. Vectors used to practice this invention include, but are notlimited to RNA, autonomous self-replicating circular or linear DNA orRNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin bp 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

Host Cells and Transformed Cells

The invention also provides a transformed, transfected, infected ortransduced cell comprising a nucleic acid sequence of the invention,e.g., a sequence encoding an amylase and/or a glucoamylase, and/or aglucanase of the invention, or a vector of the invention. The host cellmay be any of the host cells familiar to those skilled in the art,including prokaryotic cells, eukaryotic cells, such as bacterial cells,fungal cells, yeast cells, mammalian cells, insect cells, or plantcells. Exemplary bacterial cells include any species within the generaEscherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas andStaphylococcus, including, e.g., Escherichia coli, Lactococcus lactis,Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, Pseudomonasfluorescens. Exemplary yeast cells include any species of Pichia,Saccharomyces, Schizosaccharomyces, or Schwanniomyces, including Pichiapastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe.Exemplary insect cells include any species of Spodoptera or Drosophila,including Drosophila S2 and Spodoptera Sf9. Exemplary animal cellsinclude CHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. Techniques for transforming a wide variety of higherplant species are well known and described in the technical andscientific literature. See, e.g., Weising (1988) Ann. Rev. Genet.22:421-477; U.S. Pat. No. 5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets are preferred.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts and other cell linescapable of expressing proteins from a compatible vector, such as theC127, 3T3, CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding the polypeptides of the invention, or modified nucleicacids of the invention, can be reproduced by amplification.Amplification can also be used to clone or modify the nucleic acids ofthe invention. Thus, the invention provides amplification primersequence pairs for amplifying nucleic acids of the invention. One ofskill in the art can design amplification primer sequence pairs for anypart of or the full length of these sequences.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary nucleic acid of the inventionover a region of at least about 25, 30, 40, 50, 75, 100, 150, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550or more, residues. The invention provides polypeptides comprisingsequences having at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore, or complete (100%) sequence identity to an exemplary polypeptideof the invention. The extent of sequence identity (homology) may bedetermined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, can refer to two or more sequences that have, e.g., atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide oramino acid residue (sequence) identity, when compared and aligned formaximum correspondence, as measured using one any known sequencecomparison algorithm, as discussed in detail below, or by visualinspection. In alternative aspects, the invention provides nucleic acidand polypeptide sequences having substantial identity to an exemplarysequence of the invention over a region of at least about 10, 20, 30,40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000 or more residues, or a region ranging frombetween about 50 residues to the full length of the nucleic acid orpolypeptide. Nucleic acid sequences of the invention can besubstantially identical over the entire length of a polypeptide codingregion.

Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. It will beappreciated that the nucleic acid sequences as set forth herein can berepresented in the traditional single character format (see, e.g.,Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co., New York) orin any other format which records the identity of the nucleotides in asequence.

Various sequence comparison programs identified herein are used in thisaspect of the invention. Protein and/or nucleic acid sequence identities(homologies) may be evaluated using any of the variety of sequencecomparison algorithms and programs known in the art. Such algorithms andprograms include, but are not limited to, TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410,1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higginset al., Methods Enzymol. 266:383-402, 1996; Altschul et al., J. Mol.Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272,1993).

Homology or sequence identity can be measured using sequence analysissoftware (e.g., Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705). Such software matches similarsequences by assigning degrees of homology to various deletions,substitutions and other modifications. The terms “homology” and“identity” in the context of two or more nucleic acids or polypeptidesequences, refer to two or more sequences or subsequences that are thesame or have a specified percentage of amino acid residues ornucleotides that are the same when compared and aligned for maximumcorrespondence over a comparison window or designated region as measuredusing any number of sequence comparison algorithms or by manualalignment and visual inspection. For sequence comparison, one sequencecan act as a reference sequence, e.g., a sequence of the invention, towhich test sequences are compared. When using a sequence comparisonalgorithm, test and reference sequences are entered into a computer,subsequence coordinates are designated, if necessary, and sequencealgorithm program parameters are designated. Default program parameterscan be used, or alternative parameters can be designated. The sequencecomparison algorithm then calculates the percent sequence identities forthe test sequences relative to the reference sequence, based on theprogram parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the numbers of contiguous residues. For example, inalternative aspects of the invention, contiguous residues ranginganywhere from 20 to the full length of an exemplary polypeptide ornucleic acid sequence of the invention are compared to a referencesequence of the same number of contiguous positions after the twosequences are optimally aligned. If the reference sequence has therequisite sequence identity to an exemplary polypeptide or nucleic acidsequence of the invention, e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore sequence identity to a sequence of the invention, that sequence iswithin the scope of the invention. In alternative embodiments,subsequences ranging from about 20 to 600, about 50 to 200, and about100 to 150 are compared to a reference sequence of the same number ofcontiguous positions after the two sequences are optimally aligned.

Methods of alignment of sequence for comparison are well known in theart. In alternative aspects, optimal alignment of sequences forcomparison can be conducted, e.g., by the local homology algorithm ofSmith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project(Gibbs, 1995). Several genomes have been sequenced, e.g., M. genitalium(Fraser et al., 1995), M. jannaschii (Bult et al., 1996), H. influenzae(Fleischmann et al., 1995), E. coli (Blattner et al., 1997), and yeast(S. cerevisiae) (Mewes et al., 1997), and D. melanogaster (Adams et al.,2000). Significant progress has also been made in sequencing the genomesof model organism, such as mouse, C. elegans, and Arabadopsis sp.Databases containing genomic information annotated with some functionalinformation are maintained by different organization, and are accessiblevia the internet.

BLAST, BLAST 2.0 and BLAST 2.2.2 algorithms also can be used to practicethe invention. They are described, e.g., in Altschul (1977) Nuc. AcidsRes. 25:3389-3402; Altschul (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are extended in both directions alongeach sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectations (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. The BLAST algorithm also performs a statisticalanalysis of the similarity between two sequences (see, e.g., Karlin &Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873). One measure ofsimilarity provided by BLAST algorithm is the smallest sum probability(P(N)), which provides an indication of the probability by which a matchbetween two nucleotide or amino acid sequences would occur by chance.For example, a nucleic acid is considered similar to a referencessequence if the smallest sum probability in a comparison of the testnucleic acid to the reference nucleic acid is less than about 0.2, morepreferably less than about 0.01, and most preferably less than about0.001. In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”). Forexample, five specific BLAST programs can be used to perform thefollowing task: (1) BLASTP and BLAST3 compare an amino acid querysequence against a protein sequence database; (2) BLASTN compares anucleotide query sequence against a nucleotide sequence database; (3)BLASTX compares the six-frame conceptual translation products of a querynucleotide sequence (both strands) against a protein sequence database;(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and, (5) TBLASTX compares the six-frame translations of a nucleotidequery sequence against the six-frame translations of a nucleotidesequence database. The BLAST programs identify homologous sequences byidentifying similar segments, which are referred to herein as“high-scoring segment pairs,” between a query amino or nucleic acidsequence and a test sequence which is preferably obtained from a proteinor nucleic acid sequence database. High-scoring segment pairs arepreferably identified (i.e., aligned) by means of a scoring matrix, manyof which are known in the art. Preferably, the scoring matrix used isthe BLOSUM62 matrix (Gonnet et al., Science 256:1443-1445, 1992;Henikoff and Henikoff, Proteins 17:49-61, 1993). Less preferably, thePAM or PAM250 matrices may also be used (see, e.g., Schwartz andDayhoff, eds., 1978, Matrices for Detecting Distance Relationships:Atlas of Protein Sequence and Structure, Washington: National BiomedicalResearch Foundation).

In one aspect of the invention, to determine if a nucleic acid has therequisite sequence identity to be within the scope of the invention, theNCBI BLAST 2.2.2 programs is used, default options to blastp. There areabout 38 setting options in the BLAST 2.2.2 program. In this exemplaryaspect of the invention, all default values are used except for thedefault filtering setting (i.e., all parameters set to default exceptfiltering which is set to OFF); in its place a “−F F” setting is used,which disables filtering. Use of default filtering often results inKarlin-Altschul violations due to short length of sequence.

The default values used in this exemplary aspect of the invention, andto determine the values in FIG. 3, as discussed above, include:

“Filter for low complexity: ON

Word Size: 3

Matrix: Blosum62

Gap Costs: Existence: 11

Extension: 1”

Other default settings can be: filter for low complexity OFF, word sizeof 3 for protein, BLOSUM62 matrix, gap existence penalty of −11 and agap extension penalty of −1. An exemplary NCBI BLAST 2.2.2 programsetting has the “−W” option default to 0. This means that, if not set,the word size defaults to 3 for proteins and 11 for nucleotides.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, the sequence of the invention can bestored, recorded, and manipulated on any medium which can be read andaccessed by a computer. Accordingly, the invention provides computers,computer systems, computer readable mediums, computer programs productsand the like recorded or stored thereon the nucleic acid and polypeptidesequences of the invention. As used herein, the words “recorded” and“stored” refer to a process for storing information on a computermedium. A skilled artisan can readily adopt any known methods forrecording information on a computer readable medium to generatemanufactures comprising one or more of the nucleic acid and/orpolypeptide sequences of the invention.

The invention also provides computers and processors comprising computerprogram products comprising sequences of the invention; and as usedherein, the terms “computer,” “computer program” and “processor” areused in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

Another aspect of the invention is a computer readable medium havingrecorded thereon at least one nucleic acid and/or polypeptide sequenceof the invention. Computer readable media include magnetically readablemedia, optically readable media, electronically readable media andmagnetic/optical media. For example, the computer readable media may bea hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital VersatileDisk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) aswell as other types of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems, which store and manipulate the sequencesand sequence information described herein. One example of a computersystem 100 is illustrated in block diagram form in FIG. 1. As usedherein, “a computer system” refers to the hardware components, softwarecomponents, and data storage components used to analyze a nucleotide orpolypeptide sequence of the invention. The computer system 100 caninclude a processor for processing, accessing and manipulating thesequence data. The processor 105 can be any well-known type of centralprocessing unit, such as, for example, the Pentium III from IntelCorporation, or similar processor from Sun, Motorola, Compaq, AMD orInternational Business Machines. The computer system 100 is a generalpurpose system that comprises the processor 105 and one or more internaldata storage components 110 for storing data, and one or more dataretrieving devices for retrieving the data stored on the data storagecomponents. A skilled artisan can readily appreciate that any one of thecurrently available computer systems are suitable.

In one aspect, the computer system 100 includes a processor 105connected to a bus which is connected to a main memory 115 (preferablyimplemented as RAM) and one or more internal data storage devices 110,such as a hard drive and/or other computer readable media having datarecorded thereon. The computer system 100 can further include one ormore data retrieving device 118 for reading the data stored on theinternal data storage devices 110. The data retrieving device 118 mayrepresent, for example, a floppy disk drive, a compact disk drive, amagnetic tape drive, or a modem capable of connection to a remote datastorage system (e.g., via the internet) etc. In some embodiments, theinternal data storage device 110 is a removable computer readable mediumsuch as a floppy disk, a compact disk, a magnetic tape, etc. containingcontrol logic and/or data recorded thereon. The computer system 100 mayadvantageously include or be programmed by appropriate software forreading the control logic and/or the data from the data storagecomponent once inserted in the data retrieving device. The computersystem 100 includes a display 120 which is used to display output to acomputer user. It should also be noted that the computer system 100 canbe linked to other computer systems 125 a-c in a network or wide areanetwork to provide centralized access to the computer system 100.Software for accessing and processing the nucleotide or amino acidsequences of the invention can reside in main memory 115 duringexecution. In some aspects, the computer system 100 may further comprisea sequence comparison algorithm for comparing a nucleic acid sequence ofthe invention. The algorithm and sequence(s) can be stored on a computerreadable medium. A “sequence comparison algorithm” refers to one or moreprograms which are implemented (locally or remotely) on the computersystem 100 to compare a nucleotide sequence with other nucleotidesequences and/or compounds stored within a data storage means. Forexample, the sequence comparison algorithm may compare the nucleotidesequences of the invention stored on a computer readable medium toreference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user. FIG. 2 is a flow diagramillustrating one aspect of a process 200 for comparing a new nucleotideor protein sequence with a database of sequences in order to determinethe homology levels between the new sequence and the sequences in thedatabase. The database of sequences can be a private database storedwithin the computer system 100, or a public database such as GENBANKthat is available through the Internet. The process 200 begins at astart state 201 and then moves to a state 202 wherein the new sequenceto be compared is stored to a memory in a computer system 100. Asdiscussed above, the memory could be any type of memory, including RAMor an internal storage device. The process 200 then moves to a state 204wherein a database of sequences is opened for analysis and comparison.The process 200 then moves to a state 206 wherein the first sequencestored in the database is read into a memory on the computer. Acomparison is then performed at a state 210 to determine if the firstsequence is the same as the second sequence. It is important to notethat this step is not limited to performing an exact comparison betweenthe new sequence and the first sequence in the database. Well-knownmethods are known to those of skill in the art for comparing twonucleotide or protein sequences, even if they are not identical. Forexample, gaps can be introduced into one sequence in order to raise thehomology level between the two tested sequences. The parameters thatcontrol whether gaps or other features are introduced into a sequenceduring comparison are normally entered by the user of the computersystem. Once a comparison of the two sequences has been performed at thestate 210, a determination is made at a decision state 210 whether thetwo sequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200. If a determination is made that the two sequences are thesame, the process 200 moves to a state 214 wherein the name of thesequence from the database is displayed to the user. This state notifiesthe user that the sequence with the displayed name fulfills the homologyconstraints that were entered. Once the name of the stored sequence isdisplayed to the user, the process 200 moves to a decision state 218wherein a determination is made whether more sequences exist in thedatabase. If no more sequences exist in the database, then the process200 terminates at an end state 220. However, if more sequences do existin the database, then the process 200 moves to a state 224 wherein apointer is moved to the next sequence in the database so that it can becompared to the new sequence. In this manner, the new sequence isaligned and compared with every sequence in the database. It should benoted that if a determination had been made at the decision state 212that the sequences were not homologous, then the process 200 would moveimmediately to the decision state 218 in order to determine if any othersequences were available in the database for comparison. Accordingly,one aspect of the invention is a computer system comprising a processor,a data storage device having stored thereon a nucleic acid sequence ofthe invention and a sequence comparer for conducting the comparison. Thesequence comparer may indicate a homology level between the sequencescompared or identify structural motifs, or it may identify structuralmotifs in sequences which are compared to these nucleic acid codes andpolypeptide codes. FIG. 3 is a flow diagram illustrating one embodimentof a process 250 in a computer for determining whether two sequences arehomologous. The process 250 begins at a start state 252 and then movesto a state 254 wherein a first sequence to be compared is stored to amemory. The second sequence to be compared is then stored to a memory ata state 256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it can be a single letter amino acid code so thatthe first and sequence sequences can be easily compared. A determinationis then made at a decision state 264 whether the two characters are thesame. If they are the same, then the process 250 moves to a state 268wherein the next characters in the first and second sequences are read.A determination is then made whether the next characters are the same.If they are, then the process 250 continues this loop until twocharacters are not the same. If a determination is made that the nexttwo characters are not the same, the process 250 moves to a decisionstate 274 to determine whether there are any more characters eithersequence to read. If there are not any more characters to read, then theprocess 250 moves to a state 276 wherein the level of homology betweenthe first and second sequences is displayed to the user. The level ofhomology is determined by calculating the proportion of charactersbetween the sequences that were the same out of the total number ofsequences in the first sequence. Thus, if every character in a first 100nucleotide sequence aligned with an every character in a secondsequence, the homology level would be 100%.

Alternatively, the computer program can compare a reference sequence toa sequence of the invention to determine whether the sequences differ atone or more positions. The program can record the length and identity ofinserted, deleted or substituted nucleotides or amino acid residues withrespect to the sequence of either the reference or the invention. Thecomputer program may be a program which determines whether a referencesequence contains a single nucleotide polymorphism (SNP) with respect toa sequence of the invention, or, whether a sequence of the inventioncomprises a SNP of a known sequence. Thus, in some aspects, the computerprogram is a program which identifies SNPs. The method may beimplemented by the computer systems described above and the methodillustrated in FIG. 3. The method can be performed by reading a sequenceof the invention and the reference sequences through the use of thecomputer program and identifying differences with the computer program.

In other aspects the computer based system comprises an identifier foridentifying features within a nucleic acid or polypeptide of theinvention. An “identifier” refers to one or more programs whichidentifies certain features within a nucleic acid sequence. For example,an identifier may comprise a program which identifies an open readingframe (ORF) in a nucleic acid sequence. FIG. 4 is a flow diagramillustrating one aspect of an identifier process 300 for detecting thepresence of a feature in a sequence. The process 300 begins at a startstate 302 and then moves to a state 304 wherein a first sequence that isto be checked for features is stored to a memory 115 in the computersystem 100. The process 300 then moves to a state 306 wherein a databaseof sequence features is opened. Such a database would include a list ofeach feature's attributes along with the name of the feature. Forexample, a feature name could be “Initiation Codon” and the attributewould be “ATG”. Another example would be the feature name “TAATAA Box”and the feature attribute would be “TAATAA”. An example of such adatabase is produced by the University of Wisconsin Genetics ComputerGroup. Alternatively, the features may be structural polypeptide motifssuch as alpha helices, beta sheets, or functional polypeptide motifssuch as enzymatic active sites, helix-turn-helix motifs or other motifsknown to those skilled in the art. Once the database of features isopened at the state 306, the process 300 moves to a state 308 whereinthe first feature is read from the database. A comparison of theattribute of the first feature with the first sequence is then made at astate 310. A determination is then made at a decision state 316 whetherthe attribute of the feature was found in the first sequence. If theattribute was found, then the process 300 moves to a state 318 whereinthe name of the found feature is displayed to the user. The process 300then moves to a decision state 320 wherein a determination is madewhether move features exist in the database. If no more features doexist, then the process 300 terminates at an end state 324. However, ifmore features do exist in the database, then the process 300 reads thenext sequence feature at a state 326 and loops back to the state 310wherein the attribute of the next feature is compared against the firstsequence. If the feature attribute is not found in the first sequence atthe decision state 316, the process 300 moves directly to the decisionstate 320 in order to determine if any more features exist in thedatabase. Thus, in one aspect, the invention provides a computer programthat identifies open reading frames (ORFs).

A polypeptide or nucleic acid sequence of the invention can be storedand manipulated in a variety of data processor programs in a variety offormats. For example, a sequence can be stored as text in a wordprocessing file, such as MicrosoftWORD or WORDPERFECT or as an ASCIIfile in a variety of database programs familiar to those of skill in theart, such as DB2™, SYBASE™, or ORACLE™. In addition, many computerprograms and databases may be used as sequence comparison algorithms,identifiers, or sources of reference nucleotide sequences or polypeptidesequences to be compared to a nucleic acid sequence of the invention.The programs and databases used to practice the invention include, butare not limited to: MACPATTERN™ (EMBL), DiscoveryBase (MolecularApplications Group), GENEMINE™ (Molecular Applications Group), LOOK™(Molecular Applications Group), MACLOOK™ (Molecular Applications Group),BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol.Biol. 215: 403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci.USA, 85: 2444, 1988), FASTDB™ (Brutlag et al. Comp. App. Biosci.6:237-245, 1990), Catalyst (Molecular Simulations Inc.),CATALYST™/SHAPE™ (Molecular Simulations Inc.), CERIUS2.DB ACCESS™(Molecular Simulations Inc.), HYPOGEN™ (Molecular Simulations Inc.),INSIGHT II™ (Molecular Simulations Inc.), DISCOVER™ (MolecularSimulations Inc.), CHARMM™ (CHARMm™) (Molecular Simulations Inc.), Felix(Molecular Simulations Inc.), DELPHI™, (Molecular Simulations Inc.),QUANTEMM™, (Molecular Simulations Inc.), HOMOLOGY™ (MolecularSimulations Inc.), MODELER™ (Molecular Simulations Inc.), ISIS™(Molecular Simulations Inc.), QUANTA™/Protein Design (MolecularSimulations Inc.), WEBLAB™ (Molecular Simulations Inc.), WEBLABDIVERSITY EXPLORER™ (Molecular Simulations Inc.), GENE EXPLORER™(Molecular Simulations Inc.), SEQFOLD™ (Molecular Simulations Inc.), theMDL Available Chemicals Directory database, the MDL Drug Data Reportdata base, the Comprehensive Medicinal Chemistry database, Derwent'sWorld Drug Index database, the BioByteMasterFile database, the Genbankdatabase, and the Genseqn database. Many other programs and data baseswould be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices, and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites, and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated, synthetic or recombinant nucleic acidsthat hybridize under stringent conditions to an exemplary sequence ofthe invention, or a nucleic acid that encodes a polypeptide of theinvention. The stringent conditions can be highly stringent conditions,medium stringent conditions, low stringent conditions, including thehigh and reduced stringency conditions described herein. In one aspect,it is the stringency of the wash conditions that set forth theconditions which determine whether a nucleic acid is within the scope ofthe invention, as discussed below.

“Hybridization” protocols used to practice this invention includeprocesses by which a nucleic acid strand joins with a complementarystrand through base pairing. Hybridization reactions can be sensitiveand selective so that a particular sequence of interest can beidentified even in samples in which it is present at low concentrations.Stringent conditions can be defined by, for example, the concentrationsof salt or formamide in the prehybridization and hybridizationsolutions, or by the hybridization temperature, and are well known inthe art. For example, stringency can be increased by reducing theconcentration of salt, increasing the concentration of formamide, orraising the hybridization temperature, altering the time ofhybridization, as described in detail, below. In alternative aspects,nucleic acids of the invention are defined by their ability to hybridizeunder various stringency conditions (e.g., high, medium, and low), asset forth herein.

In alternative embodiments, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA, antisense or sequencesencoding antibody binding peptides (epitopes), motifs, active sites andthe like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200ug/ml sheared and denatured salmon sperm DNA). In one aspect, nucleicacids of the invention are defined by their ability to hybridize underreduced stringency conditions comprising 35% formamide at a reducedtemperature of 35° C.

Following hybridization, the filter may be washed with 6×SSC, 0.5% SDSat 50° C. These conditions are considered to be “moderate” conditionsabove 25% formamide and “low” conditions below 25% formamide. A specificexample of “moderate” hybridization conditions is when the abovehybridization is conducted at 30% formamide. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 10% formamide.

The temperature range corresponding to a particular level of stringencycan be further narrowed by calculating the purine to pyrimidine ratio ofthe nucleic acid of interest and adjusting the temperature accordingly.Nucleic acids of the invention are also defined by their ability tohybridize under high, medium, and low stringency conditions as set forthin Ausubel and Sambrook. Variations on the above ranges and conditionsare well known in the art. Hybridization conditions are discussedfurther, below.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na⁺ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention.

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with an amylaseactivity or fragments thereof or for identifying amylase genes. In oneaspect, the probe comprises at least 10 consecutive bases of a nucleicacid of the invention. Alternatively, a probe of the invention can be atleast about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about30 to 70, consecutive bases of a sequence as set forth in a nucleic acidof the invention. The probes identify a nucleic acid by binding and/orhybridization. The probes can be used in arrays of the invention, seediscussion below, including, e.g., capillary arrays. The probes of theinvention can also be used to isolate other nucleic acids orpolypeptides.

The probes of the invention can be used to determine whether abiological sample, such as a soil sample, contains an organism having anucleic acid sequence of the invention or an organism from which thenucleic acid was obtained. In such procedures, a biological samplepotentially harboring the organism from which the nucleic acid wasisolated is obtained and nucleic acids are obtained from the sample. Thenucleic acids are contacted with the probe under conditions which permitthe probe to specifically hybridize to any complementary sequencespresent in the sample. Where necessary, conditions which permit theprobe to specifically hybridize to complementary sequences may bedetermined by placing the probe in contact with complementary sequencesfrom samples known to contain the complementary sequence, as well ascontrol sequences which do not contain the complementary sequence.Hybridization conditions, such as the salt concentration of thehybridization buffer, the formamide concentration of the hybridizationbuffer, or the hybridization temperature, may be varied to identifyconditions which allow the probe to hybridize specifically tocomplementary nucleic acids (see discussion on specific hybridizationconditions).

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product. Manymethods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures, and dot blots. Protocols for each of theseprocedures are provided in Ausubel and Sambrook.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). In one aspect, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook (seediscussion on amplification reactions). In such procedures, the nucleicacids in the sample are contacted with the probes, the amplificationreaction is performed, and any resulting amplification product isdetected. The amplification product may be detected by performing gelelectrophoresis on the reaction products and staining the gel with anintercalator such as ethidium bromide. Alternatively, one or more of theprobes may be labeled with a radioactive isotope and the presence of aradioactive amplification product may be detected by autoradiographyafter gel electrophoresis.

Probes derived from sequences near the 3′ or 5′ ends of a nucleic acidsequence of the invention can also be used in chromosome walkingprocedures to identify clones containing additional, e.g., genomicsequences. Such methods allow the isolation of genes which encodeadditional proteins of interest from the host organism.

In one aspect, nucleic acid sequences of the invention are used asprobes to identify and isolate related nucleic acids. In some aspects,the so-identified related nucleic acids may be cDNAs or genomic DNAsfrom organisms other than the one from which the nucleic acid of theinvention was first isolated. In such procedures, a nucleic acid sampleis contacted with the probe under conditions which permit the probe tospecifically hybridize to related sequences. Hybridization of the probeto nucleic acids from the related organism is then detected using any ofthe methods described above.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency can vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent), and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.Hybridization can be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's, and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4-9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe can then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature (RT) in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH7.8, 1 mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash infresh 1×SET at Tm-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, Tm, is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the Tm for a particular probe. The melting temperature of the probemay be calculated using the following exemplary formulas. For probesbetween 14 and 70 nucleotides in length the melting temperature (Tm) iscalculated using the formula: Tm=81.5+16.6(log [Na+])+0.41(fractionG+C)−(600/N) where N is the length of the probe. If the hybridization iscarried out in a solution containing formamide, the melting temperaturemay be calculated using the equation: Tm=81.5+16.6 (log[Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N) where N is thelength of the probe. Prehybridization may be carried out in 6×SSC,5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured fragmented salmonsperm DNA or 6×SSC, 5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denaturedfragmented salmon sperm DNA, 50% formamide. Formulas for SSC andDenhardt's and other solutions are listed, e.g., in Sambrook.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the Tm. For shorterprobes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the Tm. In one aspect, hybridizations in6×SSC are conducted at approximately 68° C. In one aspect,hybridizations in 50% formamide containing solutions are conducted atapproximately 42° C. All of the foregoing hybridizations would beconsidered to be under conditions of high stringency.

Following hybridization, the filter is washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content), and the nucleic acid type (e.g., RNA v. DNA). Examplesof progressively higher stringency condition washes are as follows:2×SSC, 0.1% SDS at room temperature for 15 minutes (low stringency);0.1×SSC, 0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes.

Nucleic acids which have hybridized to the probe can be identified byautoradiography or other conventional techniques. The above proceduremay be modified to identify nucleic acids having decreasing levels ofhomology to the probe sequence. For example, to obtain nucleic acids ofdecreasing homology to the detectable probe, less stringent conditionsmay be used. For example, the hybridization temperature may be decreasedin increments of 5° C. from 68° C. to 42° C. in a hybridization bufferhaving a Na+ concentration of approximately 1M. Following hybridization,the filter may be washed with 2×SSC, 0.5% SDS at the temperature ofhybridization. These conditions are considered to be “moderate”conditions above 50° C. and “low” conditions below 50° C. An example of“moderate” hybridization conditions is when the above hybridization isconducted at 55° C. An example of “low stringency” hybridizationconditions is when the above hybridization is conducted at 45° C.

Alternatively, the hybridization protocols used to practice thisinvention may be carried out in buffers, such as 6×SSC, containingformamide at a temperature of 42° C. In this case, the concentration offormamide in the hybridization buffer may be reduced in 5% incrementsfrom 50% to 0% to identify clones having decreasing levels of homologyto the probe. Following hybridization, the filter may be washed with6×SSC, 0.5% SDS at 50° C. These conditions are considered to be“moderate” conditions above 25% formamide and “low” conditions below 25%formamide. A specific example of “moderate” hybridization conditions iswhen the above hybridization is conducted at 30% formamide. A specificexample of “low stringency” hybridization conditions is when the abovehybridization is conducted at 10% formamide.

These probes and methods of the invention can be used to isolate nucleicacids having a sequence with at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity (“homology”) to an exemplary nucleicacid sequence of the invention comprising at least about 10, 15, 20, 25,30, 35, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, or more consecutive basesthereof, and the sequences complementary thereto. Homology may bemeasured using an alignment algorithm, as discussed herein. For example,the homologous polynucleotides may have a coding sequence which is anaturally occurring allelic variant of one of the coding sequencesdescribed herein. Such allelic variants may have a substitution,deletion or addition of one or more nucleotides when compared to anucleic acid of the invention.

Additionally, the probes and methods of the invention can be used toisolate nucleic acids which encode polypeptides having at least about50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, sequence identity (homology)to a polypeptide of the invention comprising at least 5, 10, 15, 20, 25,30, 35, 40, 50, 75, 100, or 150 consecutive amino acids, as determinedusing a sequence alignment algorithm (e.g., such as the FASTA version3.0t78 algorithm with the default parameters, or a BLAST 2.2.2 programwith exemplary settings as set forth herein).

Inhibiting Expression of Amylase

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acid sequences of the invention, e.g., nucleicacids comprising antisense, siRNA, miRNA, ribozymes. Antisense sequencesare capable of inhibiting the transport, splicing or transcription ofamylase-encoding and glucoamylase-encoding genes. The inhibition can beeffected through the targeting of genomic DNA or messenger RNA. Thetranscription or function of targeted nucleic acid can be inhibited, forexample, by hybridization and/or cleavage. One particularly useful setof inhibitors provided by the present invention includesoligonucleotides which are able to either bind amylase gene or message,in either case preventing or inhibiting the production or function ofamylase. The association can be through sequence specific hybridization.Another useful class of inhibitors includes oligonucleotides which causeinactivation or cleavage of amylase message. The oligonucleotide canhave enzyme activity which causes such cleavage, such as ribozymes. Theoligonucleotide can be chemically modified or conjugated to an enzyme orcomposition capable of cleaving the complementary nucleic acid. A poolof many different such oligonucleotides can be screened for those withthe desired activity.

The inventions methods and compositions for inhibition of expression ofamylase, glucoamylase, glucosidase and other polysaccharide hydrolyzingenzymes can have a variety of industrial applications. For example,inhibition of glucosidase expression can slow or prevent spoilage.Spoilage can occur when polysaccharides, lipids or polypeptides, e.g.,structural polysaccharides, are enzymatically degraded. This can lead tothe deterioration, or rot, of fruits and vegetables. In one aspect, useof compositions of the invention that inhibit the expression and/oractivity of glucosidases, e.g., antibodies, antisense oligonucleotides,ribozymes and RNAi, are used to slow or prevent spoilage. Thus, in oneaspect, the invention provides methods and compositions comprisingapplication onto a plant or plant product (e.g., a fruit, seed, root,leaf, etc.) antibodies, antisense oligonucleotides, ribozymes and RNAiof the invention, e.g., to slow or prevent spoilage, or for anotherpurpose. These compositions also can be expressed by the plant (e.g., atransgenic plant) or another organism (e.g., a bacterium or othermicroorganism transformed with a glucosidase gene of the invention).

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingamylase message which can inhibit proteolytic activity by targetingmRNA. Strategies for designing antisense oligonucleotides are welldescribed in the scientific and patent literature, and the skilledartisan can design such amylase oligonucleotides using the novelreagents of the invention. For example, gene walking/RNA mappingprotocols to screen for effective antisense oligonucleotides are wellknown in the art, see, e.g., Ho (2000) Methods Enzymol. 314:168-183,describing an RNA mapping assay, which is based on standard moleculartechniques to provide an easy and reliable method for potent antisensesequence selection. See also Smith (2000) Eur. J. Pharm. Sci.11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl) glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisense amylasesequences of the invention (see, e.g., Gold (1995) J. of Biol. Chem.270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding amylase message.These ribozymes can inhibit amylase activity by, e.g., targeting mRNA.Strategies for designing ribozymes and selecting the amylase-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents of the invention. Ribozymes act by binding to atarget RNA through the target RNA binding portion of a ribozyme which isheld in close proximity to an enzymatic portion of the RNA that cleavesthe target RNA. Thus, the ribozyme recognizes and binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cleave and inactivate the target RNA. Cleavage ofa target RNA in such a manner will destroy its ability to directsynthesis of an encoded protein if the cleavage occurs in the codingsequence. After a ribozyme has bound and cleaved its RNA target, it canbe released from that RNA to bind and cleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNaseP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising an amylase enzyme sequence of theinvention (which includes both sense and antisense strands). The RNAimolecule can comprise a double-stranded RNA (dsRNA) molecule, e.g.,siRNA and/or miRNA. The RNAi molecule comprises a double-stranded RNA(dsRNA) molecule. The RNAi can inhibit expression of an amylase gene. Inone aspect, the RNAi, e.g., siRNA and/or miRNA, is about 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplexnucleotides in length. While the invention is not limited by anyparticular mechanism of action, the RNAi can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed todouble-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's of theinvention. The process may be practiced in vitro, ex vivo or in vivo. Inone aspect, the RNAi molecules of the invention can be used to generatea loss-of-function mutation in a cell, an organ or an animal. Methodsfor making and using RNAi molecules, e.g., siRNA and/or miRNA, forselectively degrade RNA are well known in the art, see, e.g., U.S. Pat.Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding an amylase. These methodscan be repeated or used in various combinations to generate amylaseshaving an altered or different activity or an altered or differentstability from that of an amylase encoded by the template nucleic acid.These methods also can be repeated or used in various combinations,e.g., to generate variations in gene/message expression, messagetranslation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly, genesite saturated mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-45′7; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye (1986) “Inhibition ofrestriction endonuclease Nci I cleavage by phosphorothioate groups andits application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application Ser. No. 09/407,800, “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLE CELLSAND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayre etal., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g., genesite saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),or a combination thereof are used to modify the nucleic acids of theinvention to generate amylases with new or altered properties (e.g.,activity under highly acidic or alkaline conditions, high temperatures,and the like). Polypeptides encoded by the modified nucleic acids can bescreened for an activity before testing for proteolytic or otheractivity. Any testing modality or protocol can be used, e.g., using acapillary array platform. See, e.g., U.S. Pat. Nos. 6,361,974;6,280,926; 5,939,250.

Saturation Mutagenesis, or, GSSM

The invention also provides methods for making enzyme using Gene SiteSaturation mutagenesis, or, GSSM, as described herein, and also in U.S.Pat. Nos. 6,171,820 and 6,579,258.

In one aspect, codon primers containing a degenerate N,N,G/T sequenceare used to introduce point mutations into a polynucleotide, e.g., anamylase or an antibody of the invention, so as to generate a set ofprogeny polypeptides in which a full range of single amino acidsubstitutions is represented at each amino acid position, e.g., an aminoacid residue in an enzyme active site or ligand binding site targeted tobe modified. These oligonucleotides can comprise a contiguous firsthomologous sequence, a degenerate N,N,G/T sequence, and, optionally, asecond homologous sequence. The downstream progeny translationalproducts from the use of such oligonucleotides include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,G/T sequence includes codons for all20 amino acids. In one aspect, one such degenerate oligonucleotide(comprised of, e.g., one degenerate N,N,G/T cassette) is used forsubjecting each original codon in a parental polynucleotide template toa full range of codon substitutions. In another aspect, at least twodegenerate cassettes are used—either in the same oligonucleotide or not,for subjecting at least two original codons in a parental polynucleotidetemplate to a full range of codon substitutions. For example, more thanone N,N,G/T sequence can be contained in one oligonucleotide tointroduce amino acid mutations at more than one site. This plurality ofN,N,G/T sequences can be directly contiguous, or separated by one ormore additional nucleotide sequence(s). In another aspect,oligonucleotides serviceable for introducing additions and deletions canbe used either alone or in combination with the codons containing anN,N,G/T sequence, to introduce any combination or permutation of aminoacid additions, deletions, and/or substitutions. In one aspect,simultaneous mutagenesis of two or more contiguous amino acid positionsis done using an oligonucleotide that contains contiguous N,N,G/Ttriplets, i.e. a degenerate (N,N,G/T)n sequence. In another aspect,degenerate cassettes having less degeneracy than the N,N,G/T sequenceare used. For example, it may be desirable in some instances to use(e.g. in an oligonucleotide) a degenerate triplet sequence comprised ofonly one N, where said N can be in the first second or third position ofthe triplet. Any other bases including any combinations and permutationsthereof can be used in the remaining two positions of the triplet.Alternatively, it may be desirable in some instances to use (e.g. in anoligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position×100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a nondegenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can optionally be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,amylases) molecules such that all 20 natural amino acids are representedat the one specific amino acid position corresponding to the codonposition mutagenized in the parental polynucleotide (other aspects useless than all 20 natural combinations). The 32-fold degenerate progenypolypeptides generated from each saturation mutagenesis reaction vesselcan be subjected to clonal amplification (e.g. cloned into a suitablehost, e.g., E. coli host, using, e.g., an expression vector) andsubjected to expression screening. When an individual progenypolypeptide is identified by screening to display a favorable change inproperty (when compared to the parental polypeptide, such as increasedproteolytic activity under alkaline or acidic conditions), it can besequenced to identify the correspondingly favorable amino acidsubstitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined—6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In another aspect, site-saturation mutagenesis can be used together withanother stochastic or non-stochastic means to vary sequence, e.g.,synthetic ligation reassembly (see below), shuffling, chimerization,recombination and other mutagenizing processes and mutagenizing agents.This invention provides for the use of any mutagenizing process(es),including saturation mutagenesis, in an iterative manner.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., amylases or antibodies of theinvention, with new or altered properties. SLR is a method of ligatingoligonucleotide fragments together non-stochastically. This methoddiffers from stochastic oligonucleotide shuffling in that the nucleicacid building blocks are not shuffled, concatenated or chimerizedrandomly, but rather are assembled non-stochastically. See, e.g., U.S.Pat. Nos. 6,773,900; 6,740,506; 6,713,282; 6,635,449; 6,605,449;6,537,776.

In one aspect, SLR comprises the following steps: (a) providing atemplate polynucleotide, wherein the template polynucleotide comprisessequence encoding a homologous gene; (b) providing a plurality ofbuilding block polynucleotides, wherein the building blockpolynucleotides are designed to cross-over reassemble with the templatepolynucleotide at a predetermined sequence, and a building blockpolynucleotide comprises a sequence that is a variant of the homologousgene and a sequence homologous to the template polynucleotide flankingthe variant sequence; (c) combining a building block polynucleotide witha template polynucleotide such that the building block polynucleotidecross-over reassembles with the template polynucleotide to generatepolynucleotides comprising homologous gene sequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are preferably shared by at leasttwo of the progenitor templates. The demarcation points can thereby beused to delineate the boundaries of oligonucleotide building blocks tobe generated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.

Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

In another aspect, the synthetic nature of the step in which thebuilding blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be optionallyremoved in an in vitro process (e.g. by mutagenesis) or in an in vivoprocess (e.g. by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

In one aspect, a nucleic acid building block is used to introduce anintron. Thus, functional introns are introduced into a man-made genemanufactured according to the methods described herein. The artificiallyintroduced intron(s) can be functional in a host cells for gene splicingmuch in the way that naturally-occurring introns serve functionally ingene splicing.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,amylases or antibodies of the invention, with new or altered properties.Optimized directed evolution is directed to the use of repeated cyclesof reductive reassortment, recombination and selection that allow forthe directed molecular evolution of nucleic acids through recombination.Optimized directed evolution allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Alternatively protocols for practicing these methods ofthe invention can be found in U.S. Pat. Nos. 6,773,900; 6,740,506;6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Pat. Nos. 6,773,900;6,740,506; 6,713,282; 6,635,449; 6,605,449; 6,537,776; 6,361,974.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB™ (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new amylase phenotype is identified, re-isolated,again modified, re-tested for activity. This process can be iterativelyrepeated until a desired phenotype is engineered. For example, an entirebiochemical anabolic or catabolic pathway can be engineered into a cell,including, e.g., starch hydrolysis activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new amylase phenotype), itcan be removed as a variable by synthesizing larger parentaloligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules can be used in methods of the inventionthat provide variants of polypeptides of the invention, e.g.,antibodies, amylases, and the like. In vivo shuffling can be performedutilizing the natural property of cells to recombine multimers. Whilerecombination in vivo has provided the major natural route to moleculardiversity, genetic recombination remains a relatively complex processthat involves 1) the recognition of homologies; 2) strand cleavage,strand invasion, and metabolic steps leading to the production ofrecombinant chiasma; and finally 3) the resolution of chiasma intodiscrete recombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In one aspect, the invention provides a method for producing a hybridpolynucleotide from at least a first polynucleotide (e.g., an amylaseand/or a glucoamylase of the invention) and a second polynucleotide(e.g., an enzyme, such as an amylase and/or a glucoamylase of theinvention or any other amylase, or, a tag or an epitope). The inventioncan be used to produce a hybrid polynucleotide by introducing at least afirst polynucleotide and a second polynucleotide which share at leastone region of partial sequence homology into a suitable host cell. Theregions of partial sequence homology promote processes which result insequence reorganization producing a hybrid polynucleotide. The term“hybrid polynucleotide”, as used herein, is any nucleotide sequencewhich results from the method of the present invention and containssequence from at least two original polynucleotide sequences. Suchhybrid polynucleotides can result from intermolecular recombinationevents which promote sequence integration between DNA molecules. Inaddition, such hybrid polynucleotides can result from intramolecularreductive reassortment processes which utilize repeated sequences toalter a nucleotide sequence within a DNA molecule.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (e.g., amylase) sequences of the invention.The invention also provides additional methods for isolating amylasesusing the nucleic acids and polypeptides of the invention. In oneaspect, the invention provides for variants of an amylase codingsequence (e.g., a gene, cDNA or message) of the invention, which can bealtered by any means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generatenucleic acids which encode polypeptides having characteristics whichenhance their value in industrial or laboratory applications. In suchprocedures, a large number of variant sequences having one or morenucleotide differences with respect to the sequence obtained from thenatural isolate are generated and characterized. These nucleotidedifferences can result in amino acid changes with respect to thepolypeptides encoded by the nucleic acids from the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl2, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids isevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/: 1 in a solution of 0.2 mM of each dNTP,2.2 mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100.2.5 units of Taq polymerase per 100:1 of reaction mixture is added andPCR is performed using the following regime: 94° C. for 60 seconds, 94°C. for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds(30-45 times) and 72° C. for 5 minutes. However, it will be appreciatedthat these parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described, e.g., inPCT Publication No. WO 91/16427.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described, e.g., in Arkin (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described,e.g., in Delegrave (1993) Biotechnology Res. 11:1548-1552. Random andsite-directed mutagenesis are described, e.g., in Arnold (1993) CurrentOpinion in Biotechnology 4:450-455.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in, e.g., U.S.Pat. Nos. 5,965,408; 5,939,250 (see also discussion, above).

The invention also provides variants of polypeptides of the invention(e.g., amylases) comprising sequences in which one or more of the aminoacid residues (e.g., of an exemplary polypeptide of the invention) aresubstituted with a conserved or non-conserved amino acid residue (e.g.,a conserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code. Conservativesubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics. Thus,polypeptides of the invention include those with conservativesubstitutions of sequences of the invention, e.g., the exemplarypolypeptides of the invention, including but not limited to thefollowing replacements: replacements of an aliphatic amino acid such asAlanine, Valine, Leucine and Isoleucine with another aliphatic aminoacid; replacement of a Serine with a Threonine or vice versa;replacement of an acidic residue such as Aspartic acid and Glutamic acidwith another acidic residue; replacement of a residue bearing an amidegroup, such as Asparagine and Glutamine, with another residue bearing anamide group; exchange of a basic residue such as Lysine and Argininewith another basic residue; and replacement of an aromatic residue suchas Phenylalanine, Tyrosine with another aromatic residue. Other variantsare those in which one or more of the amino acid residues of thepolypeptides of the invention includes a substituent group. Aconservative amino acid substitution can also comprise substituting oneamino acid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from an amylase, resulting in modification ofthe structure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for amylase activity can be removed.

Other variants within the scope of the invention are those in which thepolypeptide is associated with another compound, such as a compound toincrease the half-life of the polypeptide, for example, polyethyleneglycol.

Additional variants within the scope of the invention are those in whichadditional amino acids are fused to the polypeptide, such as a leadersequence, a secretory sequence, a proprotein sequence or a sequencewhich facilitates purification, enrichment, or stabilization of thepolypeptide.

In some aspects, the variants, fragments, derivatives and analogs of thepolypeptides of the invention retain the same biological function oractivity as the exemplary polypeptides, e.g., amylase activity, asdescribed herein. In other aspects, the variant, fragment, derivative,or analog includes a proprotein, such that the variant, fragment,derivative, or analog can be activated by cleavage of the proproteinportion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying amylase-encoding andglucoamylase-encoding nucleic acids to modify codon usage, and codonoptimized amylase-encoding and glucoamylase-encoding nucleic acids,including the exemplary SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO: 81 andSEQ ID NO: 82; these exemplary amylase-encoding andglucoamylase-encoding nucleic acids of the invention were generated asdiscussed in detail in Example 28, below, from SEQ ID NO:51, SEQ IDNO:3, SEQ ID NO: 47 and SEQ ID NO:25, respectively:

Wild-type Codon-optimized Enzyme SEQ ID NO: SEQ ID NO: Amylase 51 79Amylase 3 80 Glucoamylase 47 81 Glucoamylase 25 82

In one aspect, the invention provides methods for modifying codons in anucleic acid encoding an amylase to increase or decrease its expressionin a host cell. The invention also provides nucleic acids encoding anamylase modified to increase its expression in a host cell, amylase somodified, and methods of making the modified amylases. The methodcomprises identifying a “non-preferred” or a “less preferred” codon inamylase-encoding and glucoamylase-encoding nucleic acid and replacingone or more of these non-preferred or less preferred codons with a“preferred codon” encoding the same amino acid as the replaced codon andat least one non-preferred or less preferred codon in the nucleic acidhas been replaced by a preferred codon encoding the same amino acid. Apreferred codon is a codon over-represented in coding sequences in genesin the host cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as any speciesfrom the genus Escherichia or Pseudomonas, e.g., Escherichia coli andPseudomonas fluorescens); or gram positive bacteria, such as any speciesfrom the genus Bacillus, Streptomyces, Lactococcus, Lactobacillus, e.g.,Bacillus cereus, Lactobacillus gasseri, Lactococcus lactis, Lactococcuscremoris, Bacillus subtilis. Exemplary host cells also includeeukaryotic organisms, e.g., various yeast, such as Schizosaccharomycessp., Aspergillus sp., Hansenula sp., Kluyveromyces sp., Pichia sp. andSaccharomyces sp., including, e.g., Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris, and Kluyveromyces lactis,Hansenula polymorpha, Aspergillus niger, and mammalian cells and celllines and insect cells and cell lines. Thus, the invention also includesnucleic acids and polypeptides optimized for expression in theseorganisms and species.

For example, the codons of a nucleic acid encoding an amylase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the amylase was derived, a yeast, a fungi, a plant cell, an insectcell or a mammalian cell. Methods for optimizing codons are well knownin the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int. J.Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif 12:185-188; Narum(2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect. Immun.69:7250-7253, describing optimizing codons in mouse systems; Outchkourov(2002) Protein Expr. Purif 24:18-24, describing optimizing codons inyeast; Feng (2000) Biochemistry 39:15399-15409, describing optimizingcodons in E. coli; Humphreys (2000) Protein Expr. Purif 20:252-264,describing optimizing codon usage that affects secretion in E. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (e.g., an amylase), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides methods of making and using these transgenic non-humananimals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats and mice, comprising the nucleic acids of theinvention. These animals can be used, e.g., as in vivo models to studyamylase activity, or, as models to screen for agents that change theamylase activity in vivo. The coding sequences for the polypeptides tobe expressed in the transgenic non-human animals can be designed to beconstitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs and cows. See also, e.g.,Pollock (1999) J. Immunol. Methods 231:147-157, describing theproduction of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats. U.S. Pat. No. 6,211,428, describesmaking and using transgenic non-human mammals which express in theirbrains a nucleic acid construct comprising a DNA sequence. U.S. Pat. No.5,387,742, describes injecting cloned recombinant or synthetic DNAsequences into fertilized mouse eggs, implanting the injected eggs inpseudo-pregnant females, and growing to term transgenic mice whose cellsexpress proteins related to the pathology of Alzheimer's disease. U.S.Pat. No. 6,187,992, describes making and using a transgenic mouse whosegenome comprises a disruption of the gene encoding amyloid precursorprotein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing an amylase and/or a glucoamylase of theinvention, or, a fusion protein comprising an amylase and/or aglucoamylase of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a glucanase, mannanase, or xylanase), anexpression cassette or vector or a transfected or transformed cell ofthe invention. The invention also provides plant products, e.g., oils,seeds, leaves, extracts and the like, comprising a nucleic acid and/or apolypeptide (e.g., a glucanase, mannanase, or xylanase) of theinvention. The transgenic plant can be dicotyledonous (a dicot) ormonocotyledonous (a monocot). The invention provides transgenic plantswith a modified taste, solids content and/or texture, wherein thatmodification is generated by expressing at least one enzyme of theinvention either constitutively or selectively in the transgenic plant(or seed, or fruit, etc.), as described, e.g., in U.S. Pat. ApplicationNo. 20060195940.

The invention also provides methods of making and using these transgenicplants and seeds. The transgenic plant or plant cell expressing apolypeptide of the present invention may be constructed in accordancewith any method known in the art. See, for example, U.S. Pat. No.6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. The term “introducing” in thecontext of a polynucleotide, for example, a nucleotide construct ofinterest, is intended to mean presenting to the plant the polynucleotidein such a manner that the polynucleotide gains access to the interior ofa cell of the plant. Where more than one polynucleotide is to beintroduced, these polynucleotides can be assembled as part of a singlenucleotide construct, or as separate nucleotide constructs, and can belocated on the same or different transformation vectors. Accordingly,these polynucleotides can be introduced into the host cell of interestin a single transformation event, in separate transformation events, or,for example, in plants, as part of a breeding protocol. The methods ofthe invention do not depend on a particular method for introducing oneor more polynucleotides into a plant, only that the polynucleotide(s)gains access to the interior of at least one cell of the plant. Methodsfor introducing polynucleotides into plants are known in the artincluding, but not limited to, transient transformation methods, stabletransformation methods, and virus-mediated methods.

“Transient transformation” in the context of a polynucleotide isintended to mean that a polynucleotide is introduced into the plant anddoes not integrate into the genome of the plant.

By “stably introducing” or “stably introduced” in the context of apolynucleotide introduced into a plant is intended the introducedpolynucleotide is stably incorporated into the plant genome, and thusthe plant is stably transformed with the polynucleotide.

In alternative embodiments, “stable transformation” or “stablytransformed” is intended to mean that a polynucleotide, for example, anucleotide construct described herein, introduced into a plantintegrates into the genome of the plant and is capable of beinginherited by the progeny thereof, more particularly, by the progeny ofmultiple successive generations. Introduction into the genome of adesired plant can be such that the enzyme is regulated by endogenoustranscriptional or translational control elements. Transformationtechniques for both monocotyledons and dicotyledons are well known inthe art.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant. Nucleic acids of the invention can be used tomanipulate metabolic pathways of a plant in order to optimize or alterhost's expression of glucanase, mannanase, or xylanase. They can changeamylase, glucoamylase, glucanase, mannanase, or xylanase activity in aplant. Alternatively, an amylase, glucoamylase, glucanase, mannanase, orxylanase of the invention can be used in production of a transgenicplant to produce a compound not naturally produced by that plant. Thiscan lower production costs or create a novel product. In one embodiment,the enzyme of the invention may be expressed in such a way that theenzyme will not come in contact with it's substrate until desired. Forexample, an enzyme of the invention may be targeted and retained in theendoplasmic reticulum of a plant cell. Retention of the enzyme, in theendoplasmic reticulum of the cell, will prevent the enzyme from comingin contact with its substrate. The enzyme and substrate may then bebrought into contact through any means able to disrupt the subcellulararchitecture, such as, grinding, milling, heating, and the like. See, WO98/11235, WO 2003/18766, and WO 2005/096704, all of which are herebyincorporated by reference.

Selectable marker genes can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide.Selection markers used routinely in transformation include the nptllgene, which confers resistance to kanamycin and related antibiotics(Messing & Vierra. Gene 19: 259-268 (1982); Bevan et. al., Nature304:184-187 (1983)), the bar gene, which confers resistance to theherbicide phosphinothricin (White et. al., Nucl. Acids Res 18: 1062(1990), Spencer et. al. Theor. Appl. Genet 79: 625-631 (1990)), the hphgene, which confers resistance to the antibiotic hygromycin (Blochinger& Diggelmann, Mol Cell Biol 4: 2929-2931), the dhfr gene, which confersresistance to methatrexate (Bourouis et. al., EMBO J. 2(7): 1099-1104(1983)), the EPSPS gene, which confers resistance to glyphosate (U.S.Pat. Nos. 4,940,935 and 5,188,642),

Alternatively, transgenic plant material can be identified through apositive selection system, such as, the system utilizing themannose-6-phosphate isomerase gene, which provides the ability tometabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629).

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, optionally, marker genes into a targetexpression construct (e.g., a plasmid), along with positioning of thepromoter and the terminator sequences. This can involve transferring themodified gene into the plant through a suitable method. One or more ofthe sequences of the invention may be combined with sequences thatconfer resistance to insect, disease, drought, increase yield, improvenutritional quality of the grain, improve ethanol yield and the like.

For example, a construct may be introduced directly into the genomic DNAof the plant cell using techniques such as electroporation andmicroinjection of plant cell protoplasts, or the constructs can beintroduced directly to plant tissue using ballistic methods, such as DNAparticle bombardment. For example, see, e.g., Christou (1997) Plant Mol.Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein(1987) Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69,discussing use of particle bombardment to introduce transgenes intowheat; and Adam (1997) supra, for use of particle bombardment tointroduce YACs into plant cells. For example, Rinehart (1997) supra,used particle bombardment to generate transgenic cotton plants.Apparatus for accelerating particles is described U.S. Pat. No.5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000particle acceleration instrument; see also, John, U.S. Pat. No.5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describingparticle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springerlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803;Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32:1135-1148,discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S.Pat. No. 5,712,135, describing a process for the stable integration of aDNA comprising a gene that is functional in a cell of a cereal, or othermonocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. See, for example, Welsh J. R., Fundamentalsof Plant Genetics and Breeding, John Wiley & Sons, N Y (1981); CropBreeding, Wood D. R. (Ed.) American Society of Agronomy Madison, Wis.(1983); Mayo O., The Theory of Plant Breeding, Second Edition, ClarendonPress, Oxford (1987); Singh, D. P., Breeding for Resistance to Diseasesand Insect Pests, Springer-Verlag, NY (1986); and Wricke and Weber,Quantitative Genetics and Selection Plant Breeding, Walter de Gruyterand Co., Berlin (1986).

Since transgenic expression of the nucleic acids of the invention leadsto phenotypic changes, plants comprising the recombinant nucleic acidsof the invention can be sexually crossed with a second plant to obtain afinal product. Thus, the seed of the invention can be derived from across between two transgenic plants of the invention, or a cross betweena plant of the invention and another plant. The desired effects (e.g.,expression of the polypeptides of the invention to produce a plant inwhich flowering behavior is altered) can be enhanced when both parentalplants express the polypeptides (e.g., a glucanase, mannanase, orxylanase) of the invention. The desired effects can be passed to futureplant generations by standard propagation means.

Any plant may be used for introduction of the nucleotide of interest,including, but not limited to, corn or maize (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, such as canola, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables may include tomatoes (Lycopersicon esculentum), lettuce(e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans(Phaseolus limensis), peas (Lathyrus spp.), and members of the genusCucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis),and musk melon (C. melo). Ornamentals may include azalea (Rhododendronspp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscusrosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils(Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthuscaryophyllus), poinsettia (Euphorbia pulcherrima), canna (Cannaceaespp.) and chrysanthemum. Conifers that may be employed, including, forexample, pines such as loblolly pine (Pinus taeda), slash pine (Pinuselliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinuscontorta), and Monterey pine (Pinus radiata), Douglas-fir (Pseudotsugamenziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Piceaglauca); redwood (Sequoia sempervirens); true firs such as silver fir(Abies amabilis) and balsam fir (Abies balsamea); and cedars such asWestern red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparisnootkatensis). Leguminous plants may include, but are not limited to,beans and peas. Beans may include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc. Legumes may include, but are not limited to, Arachis, e.g.,peanuts, Vicia, e.g., crown vetch, hairy vetch, adzuki bean, mung bean,and chickpea, Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., commonbean and lima bean, Pisum, e.g., field bean, Melilotus, e.g., clover,Medicago, e.g., alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, andfalse indigo. Forage and turf grasses may include alfalfa, switchgrass(Panicum virgatum), Miscanthus, orchard grass, tall fescue, perennialryegrass, creeping bent grass, and redtop.

Plants of particular interest may include crop plants and plants used toproduce energy or fuel, for example, maize, alfalfa, sunflower,Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, oat, rye,millet, barley, rice, conifers, grasses, e.g., switch grass andMiscanthus, legume crops, e.g., pea, bean and soybean, starchytuber/roots, e.g., potato, sweet potato, cassava, taro, canna, sugarbeet, sugar cane and the like.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants which contain fiber cells, including, e.g., cotton,silk cotton tree (Kapok, Ceiba pentandra), desert willow, creosote bush,winterfat, balsa, ramie, kenaf, hemp, roselle, jute, sisal abaca andflax. In alternative embodiments, the transgenic plants of the inventioncan be members of the genus Gossypium, including members of anyGossypium species, such as G. arboreum; G. herbaceum, G. barbadense, andG. hirsutum.

The invention also provides transgenic plants to be used for producinglarge amounts of the polypeptides (e.g., a glucanase, mannanase, orxylanase or antibody) of the invention. For example, see Palmgren (1997)Trends Genet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producinghuman milk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants of theinvention by detecting the increase or decrease of transgene mRNA orprotein in transgenic plants. Means for detecting and quantitation ofmRNAs or proteins are well known in the art.

Polypeptides and Peptides

In one aspect, the invention provides isolated, synthetic or recombinantpolypeptides having a sequence identity (e.g., at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity) to an exemplary sequence of the invention, e.g., SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76 and/or SEQ ID NO:78, and subsequences thereof and variantsthereof. The sequence identity (homology) can be over the full length ofthe polypeptide, or, the identity can be over a region of at least about10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 ormore residues.

In one embodiment, the polypeptides of the invention can catalyze thehydrolysis of polysaccharides comprising glucose monomers, such asstarch (a polymer of glucose monomers joined by 1,4-alpha or 1,6-alphalinkages). In one aspect, the polypeptide has an amylase activity, e.g.,an alpha amylase activity, endoamylase activity, or a glucoamylaseactivity; and the term “amylase” as used herein also includes enzymeactivity which catalyzes the hydrolysis of a polysaccharide,oligosaccharide or starch. Amylases and/or glucoamylases of theinvention include polypeptides having an alpha-amylase activity, aβ-amylase activity, a glucoamylase activity, a 1,4-alpha-D-glucanglucohydrolase activity, an exoamylase activity, a glucanalpha-maltotetrahydrolase activity, a maltase activity, an isomaltaseactivity, a glucan 1, 4, alpha-glucosidase activity, analpha-glucosidase activity, a sucrase activity or an agarase activity(e.g., a β-agarase activity). For example, an amylase activity of theinvention includes alpha-amylase activity, including the ability tohydrolyze internal alpha-1,4-glucosidic linkages in starch to producesmaller molecular weight malto-dextrins. In one aspect, thealpha-amylase activity includes hydrolyzing internalalpha-1,4-glucosidic linkages in starch at random. An amylase activityof the invention includes polypeptides having glucoamylase activity,such as the ability to hydrolase glucose polymers linked by alpha-1,4-and alpha-1,6-glucosidic bonds. In one aspect, the polypeptides of theinvention have glucoamylase activity, hydrolyzing internalalpha-1,4-glucosidic linkages to yield smaller molecular weightmalto-dextrins. An amylase activity of the invention also includesglucan 1,4-alpha-glucosidase activity, or, 1,4-alpha-D-glucanglucohydrolase, commonly called glucoamylase but also calledamyloglucosidase and γ-amylase that, in one aspect, releases β-D-glucosefrom 1,4-alpha-, 1,6-alpha- and 1,3-alpha-linked glucans. An amylaseactivity of the invention also includes exo-amylase activity.

In one embodiment, the polypeptides of the invention can be used togenerate an antibody that specifically binds to (is specific for) apolypeptide of the invention, e.g., an exemplary enzyme of the invention(e.g., SEQ ID NO:2, SEQ ID NO:4, etc.).

In one aspect, the glucoamylase activity of a polypeptide of theinvention comprises catalysis of the hydrolysis of glucosidic bonds. Theglucoamylase activity can comprise catalyzing the step-wise hydrolyticrelease of D-glucose from the non-reducing ends of starch or otherrelated dextrins. The glucoamylase activity can comprise a1,4-alpha-D-glucan glucohydralase activity. The glucoamylase activitycan comprise catalysis of the hydrolysis of malto-dextrins resulting inthe generation of free glucose. The glucoamylase activity can comprisean exoamylase activity. The glucoamylase activity can comprise analpha-amylase or a β-amylase activity. The hydrolyzed glucosidic bondscan comprise alpha-1,4-glucosidic bonds or alpha-1,6-glucosidic bonds.The glucoamylase activity can comprise hydrolyzing glucosidic bonds in apolysaccharide, oligosaccharide or starch. The glucoamylase activity canfurther comprise hydrolyzing glucosidic bonds in the starch to producemaltodextrines. The glucoamylase activity can comprise cleaving amaltose or a D-glucose unit from non-reducing end of the polysaccharide,oligosaccharide or starch.

In one aspect, the invention provides alpha-amylases (alpha-amylases)that are endo-acting enzymes that can hydrolyze starch, a polymer ofglucose monomers joined by 1,4-alpha or 1,6-alpha linkages, to shortmaltodextrins. In one aspect, the invention provides glucoamylases thatare exo-acting hydrolases that can release beta-D-glucose from thenon-reducing ends of starch and related saccharides. Amylases andglucoamylases of this invention can be used commercially to liquefy andsaccharify starch during ethanol production using processes such as thedry milling process. In one aspect of a process of the invention, thedry milling process ground whole corn (the mash) is subjected to anelevated temperature (to promote gelatinization of starch) andhydrolyzed by one or more thermostable amylases, including at least oneenzyme of the invention, resulting in polysaccharide, e.g., starch,liquefaction. In one aspect, the hydrolyzed polysaccharide, e.g.,starch, is further digested by a glucoamylase of the invention, whichcan be added after the mash temperature cools down; and in one aspect,the glucose released from the polysaccharide, e.g., starch, is fermentedto ethanol by yeast added at the conclusion of the polysaccharide, e.g.,starch, hydrolysis process, or during saccharification of thepolysaccharide, e.g., starch.

In one aspect, glucosidases (e.g., glucoamylases, alpha glucosidases) ofthe invention hydrolyze internal polysaccharide bonds, e.g., alpha-1,4-and 1,6-glucosidic bonds in a polysaccharide, oligosaccharide or starch,to produce smaller molecular weight maltodextrines. In one aspect, thishydrolysis is largely at random. Thus, the invention provides methodsfor producing smaller molecular weight maltodextrines. Glucosidases ofthe invention can be used in laboratory and industrial settings tohydrolyze a polysaccharide, oligosaccharide or starch, or anymaltodextrine-comprising compound for a variety of purposes. Theseglucosidases can be used alone to provide specific hydrolysis or can becombined with other glucosidases to provide a “cocktail” with a broadspectrum of activity. Exemplary uses include the removal or partial orcomplete hydrolysis of a polysaccharide, oligosaccharide or starch, orany maltodextrine-comprising compound from biological, food, animalfeed, pharmaceutical or industrial samples.

For example, the glucosidases (e.g., glucoamylases) of the invention canbe formulated in laundry detergents to aid in the removal ofpolysaccharide-comprising, e.g., starch-containing, stains. Glucosidasesof the invention can be used as cleaning agents in detergent matrices(see industrial applications below). The glucosidases of the inventioncan be used in the initial stages (liquefaction) of polysaccharide,e.g., starch, processing, in wet corn milling, in alcohol production, inthe textile industry for starch desizing, in baking applications, in thebeverage industry, in oilfields in drilling processes; in inking ofrecycled paper; and in animal feed.

Glucosidases of the invention (e.g., glucoamylases) can have aglucosidase activity under various conditions, e.g., extremes in pHand/or temperature, oxidizing agents, and the like. The inventionprovides methods leading to alternative glucosidase preparations withdifferent catalytic efficiencies and stabilities, e.g., towardstemperature, oxidizing agents and changing wash conditions. In oneaspect, glucosidase variants can be produced using techniques ofsite-directed mutagenesis and/or random mutagenesis. In one aspect,directed evolution can be used to produce a great variety of glucosidasevariants with alternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify amylase and/or glucoamylase modulators, e.g., activators orinhibitors of amylase and/or glucoamylase activity. Briefly, testsamples (compounds, broths, extracts, and the like) are added to amylaseand/or glucoamylase assays to determine their ability to inhibitsubstrate cleavage. Inhibitors identified in this way can be used inindustry and research to reduce or prevent undesired proteolysis. Aswith amylase and/or glucoamylase, inhibitors can be combined to increasethe spectrum of activity.

An amylase and/or glucoamylase activity of the invention also includeshydrolyzing a polysaccharide, oligosaccharide or starch, at hightemperatures, low temperatures, alkaline pHs and at acidic pHs. Forexample, in one aspect, the invention provides polypeptides, and nucleicacids encoding them, having an amylase and/or glucoamylase activitywhich is thermostable. The polypeptide can retain an amylase and/orglucoamylase activity under conditions comprising a temperature range ofbetween about 37° C. to about 95° C.; between about 55° C. to about 85°C., between about 70° C. to about 95° C., or, between about 90° C. toabout 95° C. In another aspect, a polypeptide of the invention can havea amylase and/or glucoamylase activity which is thermotolerant. Thepolypeptide can retain an amylase and/or glucoamylase activity afterexposure to a temperature in the range from greater than 37° C. to about95° C. or anywhere in the range from greater than 55° C. to about 85° C.In one aspect, the polypeptide retains an amylase activity afterexposure to a temperature in the range from greater than 90° C. to about95° C. at pH 4.5.

The invention provides “amino acids” or “amino acid sequences of theinvention”, including an oligopeptide, peptide, polypeptide, or proteinsequence, or to a fragment, portion, or subunit of any of these, and tonaturally occurring, recombinant or synthetic molecules. The terms“polypeptide” and “protein” include amino acids joined to each other bypeptide bonds or modified peptide bonds, i.e., peptide isosteres, ornon-peptide bonds (synthetic bonds, synthetic polypeptides) and maycontain modified amino acids other than the 20 gene-encoded amino acids.The term “polypeptide” also includes peptides and polypeptide fragments,motifs and the like. The term also includes glycosylated polypeptides.The peptides and polypeptides of the invention also include all“mimetic” and “peptidomimetic” forms, as described in further detail,below.

The term “isolated” includes a material removed from its originalenvironment, e.g., the natural environment if it is naturally occurring.For example, a naturally occurring polynucleotide or polypeptide presentin a living animal is not isolated, but the same polynucleotide orpolypeptide, separated from some or all of the coexisting materials inthe natural system, is isolated. Such polynucleotides could be part of avector and/or such polynucleotides or polypeptides could be part of acomposition, and still be isolated in that such vector or composition isnot part of its natural environment. As used herein, an isolatedmaterial or composition can also be a “purified” composition, i.e., itdoes not require absolute purity; rather, it is intended as a relativedefinition. Individual nucleic acids obtained from a library can beconventionally purified to electrophoretic homogeneity. In alternativeaspects, the invention provides nucleic acids which have been purifiedfrom genomic DNA or from other sequences in a library or otherenvironment by at least one, two, three, four, five or more orders ofmagnitude.

The invention also provides “amylase variants” and “glucoamylasevariants” which can comprise an amino acid sequence which is derivedfrom the amino acid sequence of a “precursor amylase”, e.g., in oneaspect, an exemplary sequence of the invention (e.g., SEQ ID NO:2, SEQID NO:4, etc., or any polypeptide of this invention). The precursorglycoamylase or amylase also can include naturally-occurringglucoamylases or amylases and recombinant amylases. The amino acidsequence of the glucoamylase or amylase variant can be “derived” fromthe precursor glucoamylase or amylase amino acid sequence by thesubstitution, deletion or insertion of one or more amino acids of theprecursor amino acid sequence. Such modification can be of the“precursor DNA sequence” which encodes the amino acid sequence of theprecursor amylase rather than manipulation of the precursor amylaseenzyme per se. Suitable methods for such manipulation of the precursorDNA sequence include methods disclosed herein, as well as methods knownto those skilled in the art.

Activities of exemplary sequences of the invention are listed in thetable (“Table 1”) immediately below. To aid in reading the table, forexample, in the first row, SEQ ID NO:1, 2, represent the exemplarypolypeptide of the invention having a sequence as set forth in SEQ IDNO:2, encoded by, e.g., SEQ ID NO:1; and this exemplary sequence wasinitially isolated from Cochliobolus heterostrophus, ATCC 48331; nosignal sequence is predicted (but under certain cell in vivo conditions,the sequence may have a signal sequence); the polypeptide has“glycosidase” activity, which can be more specifically designated“amylase” enzyme activity; and the corresponding “EC” number for amylaseenzymes (an EC number is the number assigned to a type of enzymeaccording to a scheme of standardized enzyme nomenclature developed bythe Enzyme Commission of the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology, or IUBMB); “CMB20”designating “carbohydrate binding domain”; and the last column indicatesa “genetic source”, or the source of the exemplary sequence asdetermined by homology analysis of the 18/16S RNA of the cell from whichit was initially isolated. In the second row, SEQ ID NO:11, 12,represent the exemplary polypeptide of the invention having a sequenceas set forth in SEQ ID NO:12, encoded by, e.g., SEQ ID NO:11; and thisexemplary sequence was initially isolated from an unknown source; the“SS site” designates the amino terminal resides that are the signalsequence, and for SEQ ID NO:12 it is the amino terminal 21 amino acidresidues that make up the signal sequence (or, MFNQVLYGLAATALWQGQVVA,i.e., residues 1 to 21 of SEQ ID NO:12); and the polypeptide has“glycosidase” activity, which can be more specifically designated as a“glycoamylase” enzyme activity; this enzyme having an EC number of3.2.1.3; with “carbohydrate binding domain”; and a genetic match of the18/16S RNA of the cell from which it was initially isolated is Fusariumequiseti.

TABLE 1 An SEQ Exemplary Source (as ID SS General Specific ECdetermined by NO: Source site Signal Sequence Activity Activity NumberCBM20 18/16S RNA) 1, Cochliobolus Glyco- amylase 3.2.1.1 CBM20Cochliobolus 2 heterostrophus sidase heterostrophus ATCC 48331 11,Unknown AA 1- MFNQVLYGLAATALWQGQVVA Glyco- gluco- 3.2.1.3 CBM20 Fusarium12 21 sidase amylase equiseti 100% 13, Fusarium AA 1-MFTQILYGLTALSALQGQVTA Glyco- gluco- 3.2.1.3 CBM20 Fusarium 14verticillioides 21 sidase amylase verticillioides GZ3639 GZ3639 15,Cochliobolus AA 1- MLSKILLPVVALAASANAHG Glyco- gluco- CBM20 Cochliobolus16 heterostrophus 20 sidase amylase heterostrophus ATCC 48332 17,Fusarium Glyco- gluco- 3.2.1.3 CBM20 Fusarium 18 verticillioides sidaseamylase verticillioides GZ3639 GZ3639 19, Unknown AA 1-MLTLNVLTALLAPIVLSSALPA Glyco- gluco- 3.2.1.3 no Penicillium 20 22 sidaseamylase chrysogenum 100% 21, Unknown AA 1- MVLARLAWLAGLVSTAVA Glyco-amylase 3.2.1.1 no Penicillium 22 18 sidase expansum 99% 23, UnknownAA 1- MKLSHTLTALLLPLICTVSA Glyco- amylase 3.2.1.1 no Penicillium 24 20sidase chrysogenum 100% 25, Unknown AA 1- MTISRLSSVLFALALGQSALA Glyco-gluco- 3.2.1.3 CBM20 Penicillium 26 21 sidase amylase verruculosum 100%27, Unknown AA 1- MYILSSAFLLGSLALQSVLG Glyco- gluco- 3.2.1.3 CBM20Fusarium 28 20 sidase amylase merismoides 99% 29, Unknown AA 1-MLFSSLLRALSASLLAGAVQG Glyco- gluco- 3.2.1.3 CBM20 Phoma 30 21 sidaseamylase herbarum 99 3, Cochliobolus AA 1- MLLLNIFTTLFFYITCIVSA Glyco-amylase 3.2.1.1 CBM20 Cochliobolus 4 heterostrophus 20 sidaseheterostrophus ATCC 48331 31, Unknown AA1- MVLARLAWLAGLVSTAIA Glyco-amylase 3.2.1.1 no Penicillium 32 18 sidase chlysogenum 100% 33, UnknownAA1- MVGFNILTLALLAPAALS Glyco- gluco- 3.2.1.3 no Penicillium 34 18sidase amylase herquei 99% 35, Unknown AA 1- MAPRFWTTLCALTLGSAALA Glyco-gluco- 3.2.1.3 CBM20 Fusarium 36 20 sidase amylase oxysporum 100% 37,Unknown AA 1- MAPRFWIALWALTFGQAIA Glyco- gluco- 3.2.1.3 CBM20 Cordyceps38 19 sidase amylase ophioglossoides 99% 39, Unknown AA1-MAPRFWTALWALTLGHAVVA Glyco- gluco- 3.2.1.3 CBM20 Penicillium 40 20sidase amylase chlysogenum 100% 41, Unknown Glyco- gluco- 3.2.1.3 noCucurbitaria 42 sidase amylase berberidis 98% 43, Cochliobolus AA 1-MTHTSFVQASTVLSSLLALTAGQ Glyco- α-gluco- Cochliobolus 44 heterostrophus23 sidase sidase heterostrophus ATCC 48332 45, Unknown AA 1-MKLLQLAALVASLSPFTNA Glyco- amylase 3.2.1.1 no Fusarium 46 19 sidaseequiseti 100% 47, Unknown AA 1- MTRILTLALHGLALVQSVVG Glyco- gluco-3.2.1.3 CBM20 Aspergillus 48 20 sidase amylase versicolor 99 49,Aspergillus AA 1- MSFFLSCLYLSLCGSALA Glyco- amylase 3.2.1.1 noAspergillus 50 terreus 18 sidase terreus 5, Cochliobolus Glyco- α-gluco-Cochliobolus 6 heterostrophus sidase sidase heterostrophus ATCC 4833151, Aspergillus AA 1- MKWTFSLLLLLSVFGQATHA Glyco- amylase 3.2.1.1 CBM20Aspergillus 52 terreus 20 sidase terreus 53, Aspergillus AA 1-MKLSRALTVFLLHLTSTALA Glyco- amylase 3.2.1.1 no Aspergillus 54 terreus 20sidase terreus 55, Unknown AA 1- MLKQFTKRLITLTSLLALVLVAP Glyco- 3.2.1.156 27 LASA sidase 57, Unknown Glyco- 3.2.1.10 58 sidase 59, UnknownGlyco- 3.2.1.10 60 sidase 61, Unknown AA 1- MVAGFGLYGAALLTPMAAQA Glyco-3.2.1.1 62 20 sidase 63, Unknown Glyco- 3.2.1.20 64 sidase 65, UnknownAA 1- MKLKYLALVLLAVASIGLLSTPV Glyco- 3.2.1.1 66 25 GA sidase 67, UnknownAA 1- MKKNTISALVAGMVLGFASNAMA Glyco- 3.2.1.1 68 23 sidase 69, UnknownAA1- MNRPGTGASGRPQSRSATSWQS Glyco- 3.2.1.1 70 44 RNGGWLLASLLAVCFATAPVRAsidase 7, Cochliobolus Glyco- gluco- 3.2.1.3 no Cochliobolus 8heterostrophus sidase amylase heterostrophus ATCC 48331 71, UnknownGlyco- 3.2.1.20 72 sidase 73, Thermomyces Glyco- 3.2.1.3 74 lanuginosussidase ATCC 200065 75, Unknown AA 1- Glyco- amylase 3.2.1.1 76 24 sidase9, Unknown AA 1- MLTLNVLTALLAPGVLSSALPA Glyco- gluco- 3.2.1.3 noPenicillium 10 22 sidase amylase expansum 99%

Polypeptides of the invention can also be shorter than the full lengthof exemplary polypeptides. In alternative aspects, the inventionprovides polypeptides (peptides, fragments) ranging in size betweenabout 5 and the full length of a polypeptide, e.g., an enzyme, such asan amylase; exemplary sizes being of about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 175, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or more residues,e.g., contiguous residues of an exemplary amylase and/or glucoamylase ofthe invention. Peptides of the invention can be useful as, e.g.,labeling probes, antigens, toleragens, motifs, amylase active sites.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked. Glycosylationcan be added to any polypeptide of the invention to generate an enzymethat is more thermotolerant or thermostable than the “parent” enzyme (towhich the glycosylation was added). The glycosylation can be added byeither chemical or by cellular biosynthetic mechanisms.

The invention provides amylases having a broad range of specificactivity over a broad range of temperatures, e.g., at about 37° C. inthe range from about 10 to 10,000, or, 100 to about 1000 units permilligram of protein. Amylases and/or glucoamylases of the invention canalso have activity at temperatures as high as 120° C. In alternativeaspects, the amylase used in these methods is active at thesetemperatures, e.g., active at temperatures in a range of between about80° C. to about 115° C., between about 100° C. to about 110° C., andfrom about 105° C. to about 108° C. However, amylases and/orglucoamylases of the invention can also have activity at lowtemperatures, e.g., as low as 4° C. to 5° C.

The Tm of an enzyme of the invention can be shifted (for example, can beshifted between about 10° C. to 90° C.) by heat activation. For example,the Tm of SEQ ID NO:336/337 can be shifted about 17° C. to 87° C. byheat activation: for example, 80° C. preincubation for 5 minutes.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has anamylase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components (e.g., they can becompletely or partially synthetic, or “mimetic”). In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The invention provides novel glucoamylases and amylases (e.g., alphaamylases), including the exemplary enzymes of the invention, nucleicacids encoding them, antibodies that bind them, and methods for makingand using them. In one aspect, the polypeptides of the invention have anamylase and/or glucoamylase activity, as described herein, including,e.g., the ability to hydrolyze polysaccharides, oligosaccharides and/orstarches, into sugars. In one aspect, the polypeptides of the inventionhave a glucoamylase or an amylase (e.g., alpha amylase) activity. Inalternative aspects, the amylases and/or glucoamylases of the inventionhave activities that have been modified from those of the exemplaryamylases and/or glucoamylases described herein.

The invention includes amylases and/or glucoamylases of the inventionwith and without signal sequences (including signal sequences of theinvention, see e.g., Table 1, or other signal sequences) and the signalsequences themselves (e.g., Table 1). The invention also includepolypeptides (e.g., fusion proteins) comprising a signal sequence of theinvention, see, e.g., Table 1. The polypeptide comprising a signalsequence of the invention can be an amylase and/or a glucoamylase of theinvention or another amylase or another enzyme or other polypeptide.

The invention includes immobilized amylases, glucoamylases,anti-glucoamylase, anti-amylase antibodies and fragments thereof. Theinvention provides methods for inhibiting amylase and/or glucoamylaseactivity, e.g., using dominant negative mutants or anti-amylase oranti-glucoamylase antibodies of the invention. The invention includesheterocomplexes, e.g., fusion proteins, heterodimers, etc., comprisingthe amylases and/or glucoamylases of the invention.

In one aspect, amylases (e.g., alpha amylases) and/or glucoamylases ofthe invention hydrolyze internal polysaccharide or oligosaccharidebonds, e.g., alpha-1,4- and 1,6-glucosidic bonds in starch to producesmaller molecular weight maltodextrines. In one aspect, this hydrolysisis largely at random. Thus, the invention provides methods for producingsmaller molecular weight maltodextrines.

Amylases and/or glucoamylases of the invention can be used in laboratoryand industrial settings to hydrolyze polysaccharide, oligosaccharide orstarch or any maltodextrine-comprising compound for a variety ofpurposes. These amylases and/or glucoamylases can be used alone toprovide specific hydrolysis or can be combined with other amylases toprovide a “cocktail” with a broad spectrum of activity. Exemplary usesinclude the removal or partial or complete hydrolysis of polysaccharide,oligosaccharide or starch or any maltodextrine-comprising compound frombiological, food, animal feed, pharmaceutical or industrial samples.

For example, the amylases of the present invention can be formulated inlaundry detergents to aid in the removal of polysaccharide-comprising,e.g., starch-containing, stains. In one aspect, the invention providesdetergents comprising amylases and/or glucoamylases of the invention,including amylases and/or glucoamylases active under alkalineconditions, and methods of making and using them. These detergentcompositions include laundry and dishwashing (e.g., autodishwashing)solutions and application. Amylases and/or glucoamylases of theinvention can be used as cleaning agents in any detergent matrices (seeindustrial applications below). The amylases and/or glucoamylases of thepresent invention can be used in the initial stages (liquefaction) ofpolysaccharide, e.g., starch, processing, in wet corn milling, inalcohol production, in the textile industry for polysaccharide, e.g.,starch, desizing, in baking applications, in the beverage industry, inoilfields in drilling processes; in inking of recycled paper; and inanimal feed.

Amylases and/or glucoamylases of the invention can have an amylaseactivity under various conditions, e.g., extremes in pH and/ortemperature, oxidizing agents, and the like. The invention providesmethods leading to alternative amylase preparations with differentcatalytic efficiencies and stabilities, e.g., towards temperature,oxidizing agents and changing wash conditions. In one aspect, amylasevariants can be produced using techniques of site-directed mutagenesisand/or random mutagenesis. In one aspect, directed evolution can be usedto produce a great variety of amylase variants with alternativespecificities and stability.

The invention also provides methods of discovering new amylases and/orglucoamylases using the nucleic acids, polypeptides and antibodies ofthe invention. In one aspect, lambda phage libraries are screened forexpression-based discovery of amylases. In one aspect, the inventionuses lambda phage libraries in screening to allow detection of toxicclones; improved access to substrate; reduced need for engineering ahost, by-passing the potential for any bias resulting from mass excisionof the library; and, faster growth at low clone densities. Screening oflambda phage libraries can be in liquid phase or in solid phase. In oneaspect, the invention provides screening in liquid phase. This gives agreater flexibility in assay conditions; additional substrateflexibility; higher sensitivity for weak clones; and ease of automationover solid phase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

The present invention includes amylase and/or glucoamylase enzymes whichare non-naturally occurring carbonyl hydrolase variants (e.g., amylaseand/or glucoamylase variants) having a different proteolytic activity,stability, substrate specificity, pH profile and/or performancecharacteristic as compared to the precursor carbonyl hydrolase fromwhich the amino acid sequence of the variant is derived. Specifically,such amylase variants have an amino acid sequence not found in nature,which is derived by substitution of a plurality of amino acid residuesof a precursor amylase and/or glucoamylase with different amino acids.The precursor amylase and/or glucoamylase may be a naturally-occurringamylase or a recombinant amylase. The useful amylase and/or glucoamylasevariants encompass the substitution of any of the naturally occurringL-amino acids at the designated amino acid residue positions.

Amylase and Glucoamylase Signal Sequences

The invention provides signal sequences consisting of or comprising apeptide having a sequence comprising residues 1 to 12, 1 to 13, 1 to 14,1 to 15, 1 to 16, 1 to 17, 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22,1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to 27, 1 to 28, 1 to 28, 1 to 30or 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to 35, 1 to 36, 1 to 37, 1 to38, or 1 to 39, or longer, of a polypeptide of the invention. Forexample, the invention provides amylase (e.g., alpha amylase) orglucoamylase signal sequences and nucleic acids encoding these signalsequences, e.g., exemplary peptides of the invention having sequences asset forth in the Table above.

The amylase and/or glucoamylase signal sequences of the invention can beisolated peptides, or, sequences joined to another amylase and/orglucoamylase or a non-amylase or non-glucoamylase polypeptide, e.g., asa fusion protein. In one aspect, the invention provides polypeptidescomprising amylase and/or glucoamylase signal sequences of theinvention. In one aspect, polypeptides comprising amylase and/orglucoamylase signal sequences of the invention comprise sequencesheterologous to an amylase and/or a glucoamylase of the invention (e.g.,a fusion protein comprising an amylase signal sequence of the inventionand sequences from another amylase or a non-amylase protein). In oneaspect, the invention provides amylases and/or glucoamylases of theinvention with heterologous signal sequences, e.g., sequences with ayeast signal sequence. For example, an amylase and/or a glucoamylase ofthe invention comprising a heterologous signal sequence in a vectors,e.g., a pPIC series vector (Invitrogen, Carlsbad, Calif.).

In one aspect, the signal sequences of the invention are identifiedfollowing identification of novel amylase and/or glucoamylasepolypeptides. The pathways by which proteins are sorted and transportedto their proper cellular location are often referred to as proteintargeting pathways. One of the most important elements in all of thesetargeting systems is a short amino acid sequence at the amino terminusof a newly synthesized polypeptide called the signal sequence. Thissignal sequence directs a protein to its appropriate location in thecell and is removed during transport or when the protein reaches itsfinal destination. Most lysosomal, membrane, or secreted proteins havean amino-terminal signal sequence that marks them for translocation intothe lumen of the endoplasmic reticulum. More than 100 signal sequencesfor proteins in this group have been determined. The signal sequencescan vary in length from between about 13 to 36, or anywhere from betweenabout 10 to 40, amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel amylase signal peptides are identified by a methodreferred to as SIGNALP™. SignalP™ uses a combined neural network whichrecognizes both signal peptides and their cleavage sites; see, e.g.,Nielsen (1997) “Identification of prokaryotic and eukaryotic signalpeptides and prediction of their cleavage sites”, Protein Engineering,vol. 10, no. 1, p. 1-6.

It should be understood that in some aspects amylases and/orglucoamylases of the invention may not have signal sequences. In oneaspect, the invention provides the amylases and/or glucoamylases of theinvention lacking all or part of a signal sequence, e.g. the signalsequences of the invention (see Table 1). In one aspect, the inventionprovides a nucleic acid sequence encoding a signal sequence from oneamylase operably linked to a nucleic acid sequence of a differentamylase or, optionally, a signal sequence from a non-amylase protein maybe desired. Table 1 shows exemplary signal sequences of the invention.

Amylase and Glycoamylase Prepro and Signal Sequences and CatalyticDomains

In addition to signal sequences (e.g., signal peptides (SPs)), asdiscussed above, the invention provides prepro domains and catalyticdomains (CDs). The SPs, prepro domains and/or CDs of the invention canbe isolated, synthetic or recombinant peptides or can be part of afusion protein, e.g., as a heterologous domain in a chimeric protein.The invention provides nucleic acids encoding these catalytic domains(CDs) (e.g., “active sites”), prepro domains and signal sequences (SPs,e.g., a peptide having a sequence comprising/consisting of aminoterminal residues of a polypeptide of the invention).

The amylase and/or glucoamylase signal sequences (SPs), catalyticdomains (CDs) and/or prepro sequences of the invention can be isolatedpeptides, or, sequences joined to another amylase or a non-amylase or anon-glucoamylase polypeptide, e.g., as a fusion (chimeric) protein. Inone aspect, polypeptides comprising amylase signal sequences SPs and/orprepro of the invention comprise sequences heterologous to amylasesand/or glucoamylases of the invention (e.g., a fusion protein comprisingan SP and/or prepro of the invention and sequences from another amylaseand/or glucoamylase, or a non-amylase or a non-glucoamylase protein). Inone aspect, the invention provides amylases and/or glucoamylases of theinvention with heterologous CDs, SPs and/or prepro sequences, e.g.,sequences with a yeast signal sequence. An amylase and/or a glucoamylaseof the invention can comprise a heterologous CD, SP and/or prepro in avector, e.g., a pPIC series vector (Invitrogen, Carlsbad, Calif.).

In one aspect, SPs, CDs, and/or prepro sequences of the invention areidentified following identification of novel amylase polypeptides. Thepathways by which proteins are sorted and transported to their propercellular location are often referred to as protein targeting pathways.One of the most important elements in all of these targeting systems isa short amino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. The signal sequences can vary in length from 13to 45 or more amino acid residues. Various methods of recognition ofsignal sequences are known to those of skill in the art. For example, inone aspect, novel hydrolase signal peptides are identified by a methodreferred to as SignalP. SignalP uses a combined neural network whichrecognizes both signal peptides and their cleavage sites. (Nielsen, etal., “Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering, vol. 10, no.1, p. 1-6 (1997).

In some aspects, an amylase and/or a glucoamylase of the invention maynot have SPs and/or prepro sequences, and/or catalytic domains (CDs). Inone aspect, the invention provides amylases and/or glucoamylases lackingall or part of an SP, a CD and/or a prepro domain. In one aspect, theinvention provides a nucleic acid sequence encoding a signal sequence(SP), a CD and/or prepro from one amylase and/or glucoamylase operablylinked to a nucleic acid sequence of a different amylase and/orglucoamylase, or, optionally, a signal sequence (SPs), a CD and/orprepro domain from a non-amylase or non-glucoamylase protein may bedesired.

The invention also provides isolated, synthetic or recombinantpolypeptides comprising signal sequences (SPs), prepro domain and/orcatalytic domains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toan amylase) with an SP, prepro domain and/or CD. The sequence to whichthe SP, prepro domain and/or CD are not naturally associated can be onthe SP's, prepro domain and/or CD's amino terminal end, carboxy terminalend, and/or on both ends of the SP and/or CD. In one aspect, theinvention provides an isolated, synthetic or recombinant polypeptidecomprising (or consisting of) a polypeptide comprising a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention withthe proviso that it is not associated with any sequence to which it isnaturally associated (e.g., amylase and/or a glucoamylase sequence).Similarly in one aspect, the invention provides isolated, synthetic orrecombinant nucleic acids encoding these polypeptides. Thus, in oneaspect, the isolated, synthetic or recombinant nucleic acid of theinvention comprises coding sequence for a signal sequence (SP), preprodomain and/or catalytic domain (CD) of the invention and a heterologoussequence (i.e., a sequence not naturally associated with the a signalsequence (SP), prepro domain and/or catalytic domain (CD) of theinvention). The heterologous sequence can be on the 3′ terminal end, 5′terminal end, and/or on both ends of the SP, prepro domain and/or CDcoding sequence.

The polypeptides of the invention include amylases and/or aglucoamylases in an active or inactive form. For example, thepolypeptides of the invention include proproteins before “maturation” orprocessing of prepro sequences, e.g., by a proprotein-processing enzyme,such as a proprotein convertase to generate an “active” mature protein.The polypeptides of the invention include amylases and/or aglucoamylases inactive for other reasons, e.g., before “activation” by apost-translational processing event, e.g., an endo- or exo-peptidase orproteinase action, a phosphorylation event, an amidation, aglycosylation or a sulfation, a dimerization event, and the like.Methods for identifying “prepro” domain sequences, CDs, and signalsequences are well known in the art, see, e.g., Van de Ven (1993) Crit.Rev. Oncog. 4(2):115-136. For example, to identify a prepro sequence,the protein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The polypeptides of the invention include all active forms, includingactive subsequences, e.g., catalytic domains (CDs) or active sites, ofan enzyme of the invention. In one aspect, the invention providescatalytic domains or active sites as set forth below. In one aspect, theinvention provides a peptide or polypeptide comprising or consisting ofan active site domain as predicted through use of a database such asPfam (which is a large collection of multiple sequence alignments andhidden Markov models covering many common protein families, The Pfamprotein families database, A. Bateman, E. Birney, L. Cerruti, R. Durbin,L. Etwiller, S. R. Eddy, S. Griffiths-Jones, K. L. Howe, M. Marshall,and E. L. L. Sonnhammer, Nucleic Acids Research, 30(1):276-280, 2002) orequivalent.

Hybrid Amylases and Glucoamylases, and Peptide Libraries

In one aspect, the invention provides hybrid amylases and/or aglucoamylases and fusion proteins, including peptide libraries,comprising sequences of the invention. The peptide libraries of theinvention can be used to isolate peptide modulators (e.g., activators orinhibitors) of targets, such as amylase and/or a glucoamylasesubstrates, receptors, enzymes. The peptide libraries of the inventioncan be used to identify formal binding partners of targets, such asligands, e.g., cytokines, hormones and the like.

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of amylases and/or glucoamylases of the invention and otherpeptides, including known and random peptides. They can be fused in sucha manner that the structure of the amylases and/or a glucoamylases isnot significantly perturbed and the peptide is metabolically orstructurally conformationally stabilized. This allows the creation of apeptide library that is easily monitored both for its presence withincells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of an amylase and/or a glucoamylase sequence. In one aspect,the variants of the invention exhibit the same qualitative biologicalactivity as the naturally occurring analogue. Alternatively, thevariants can be selected for having modified characteristics. In oneaspect, while the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed amylase and/or a glucoamylasevariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, as discussed herein forexample, M13 primer mutagenesis and PCR mutagenesis. Screening of themutants can be done using assays of proteolytic activities. Inalternative aspects, amino acid substitutions can be single residues;insertions can be on the order of from about 1 to 20 amino acids,although considerably larger insertions can be done. Deletions can rangefrom about 1 to about 20, 30, 40, 50, 60, 70 residues or more. To obtaina final derivative with the optimal properties, substitutions,deletions, insertions or any combination thereof may be used. Generally,these changes are done on a few amino acids to minimize the alterationof the molecule. However, larger changes may be tolerated in certaincircumstances.

The invention provides amylases and/or a glucoamylases where thestructure of the polypeptide backbone, the secondary or the tertiarystructure, e.g., an alpha-helical or beta-sheet structure, has beenmodified. In one aspect, the charge or hydrophobicity has been modified.In one aspect, the bulk of a side chain has been modified. Substantialchanges in function or immunological identity are made by selectingsubstitutions that are less conservative. For example, substitutions canbe made which more significantly affect: the structure of thepolypeptide backbone in the area of the alteration, for example aalpha-helical or a beta-sheet structure; a charge or a hydrophobic siteof the molecule, which can be at an active site; or a side chain. Theinvention provides substitutions in polypeptide of the invention where(a) a hydrophilic residues, e.g. seryl or threonyl, is substituted for(or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl,valyl or alanyl; (b) a cysteine or proline is substituted for (or by)any other residue; (c) a residue having an electropositive side chain,e.g. lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine. The variants can exhibitthe same qualitative biological activity (i.e. amylase and/or aglucoamylase activity) although variants can be selected to modify thecharacteristics of the amylases and/or a glucoamylases as needed.

In one aspect, amylases and/or glucoamylases of the invention compriseepitopes or purification tags, signal sequences or other fusionsequences, etc. In one aspect, the Amylases and/or glucoamylases of theinvention can be fused to a random peptide to form a fusion polypeptide.By “fused” or “operably linked” herein is meant that the random peptideand the amylase and/or a glucoamylase are linked together, in such amanner as to minimize the disruption to the stability of the amylaseand/or a glucoamylase structure, e.g., it retains amylase and/or aglucoamylase activity. The fusion polypeptide (or fusion polynucleotideencoding the fusion polypeptide) can comprise further components aswell, including multiple peptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

Screening Methodologies and “On-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides for amylaseand/or a glucoamylase activity, to screen compounds as potentialmodulators, e.g., activators or inhibitors, of an amylase and/or aglucoamylase activity, for antibodies that bind to a polypeptide of theinvention, for nucleic acids that hybridize to a nucleic acid of theinvention, to screen for cells expressing a polypeptide of the inventionand the like.

Capillary Arrays

Capillary arrays, such as the GIGAMATRIX™, Diversa Corporation, SanDiego, Calif., can be used to in the methods of the invention. Nucleicacids or polypeptides of the invention can be immobilized to or appliedto an array, including capillary arrays. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. Capillary arrays provide another system for holding andscreening samples. For example, a sample screening apparatus can includea plurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The apparatus can further include interstitialmaterial disposed between adjacent capillaries in the array, and one ormore reference indicia formed within of the interstitial material. Acapillary for screening a sample, wherein the capillary is adapted forbeing bound in an array of capillaries, can include a first walldefining a lumen for retaining the sample, and a second wall formed of afiltering material, for filtering excitation energy provided to thelumen to excite the sample.

A polypeptide or nucleic acid, e.g., a ligand, can be introduced into afirst component into at least a portion of a capillary of a capillaryarray. Each capillary of the capillary array can comprise at least onewall defining a lumen for retaining the first component. An air bubblecan be introduced into the capillary behind the first component. Asecond component can be introduced into the capillary, wherein thesecond component is separated from the first component by the airbubble. A sample of interest can be introduced as a first liquid labeledwith a detectable particle into a capillary of a capillary array,wherein each capillary of the capillary array comprises at least onewall defining a lumen for retaining the first liquid and the detectableparticle, and wherein the at least one wall is coated with a bindingmaterial for binding the detectable particle to the at least one wall.The method can further include removing the first liquid from thecapillary tube, wherein the bound detectable particle is maintainedwithin the capillary, and introducing a second liquid into the capillarytube.

The capillary array can include a plurality of individual capillariescomprising at least one outer wall defining a lumen. The outer wall ofthe capillary can be one or more walls fused together. Similarly, thewall can define a lumen that is cylindrical, square, hexagonal or anyother geometric shape so long as the walls form a lumen for retention ofa liquid or sample. The capillaries of the capillary array can be heldtogether in close proximity to form a planar structure. The capillariescan be bound together, by being fused (e.g., where the capillaries aremade of glass), glued, bonded, or clamped side-by-side. The capillaryarray can be formed of any number of individual capillaries, forexample, a range from 100 to 4,000,000 capillaries. A capillary arraycan form a micro titer plate having about 100,000 or more individualcapillaries bound together.

Arrays, or “Biochips”

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array, e.g., an “array” or “microarray” or “biochip” or“chip”, which in one embodiment comprises a plurality of targetelements, each target element comprising a defined amount of one or morepolypeptides (including antibodies) or nucleic acids immobilized onto adefined area of a substrate surface, where at least one of the “targetelements” is a polypeptide (e.g., an enzyme or antibody) of theinvention, or a nucleic acid of the invention.

Arrays of the invention can be used to screen for or monitor librariesof compositions (e.g., small molecules, antibodies, nucleic acids, etc.)for their ability to bind to or modulate the activity of a nucleic acidor a polypeptide of the invention. For example, in one aspect of theinvention, a monitored parameter is transcript expression of an amylaseand/or a glucoamylase gene. One or more, or, all the transcripts of acell can be measured by hybridization of a sample comprising transcriptsof the cell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array, or “biochip.” By using an “array” of nucleic acids on amicrochip, some or all of the transcripts of a cell can besimultaneously quantified. Alternatively, arrays comprising genomicnucleic acid can also be used to determine the genotype of a newlyengineered strain made by the methods of the invention. Polypeptidearrays” can also be used to simultaneously quantify a plurality ofproteins. The present invention can be practiced with any known “array,”also referred to as a “microarray” or “nucleic acid array” or“polypeptide array” or “antibody array” or “biochip,” or variationthereof. Arrays are generically a plurality of “spots” or “targetelements,” each target element comprising a defined amount of one ormore biological molecules, e.g., oligonucleotides, immobilized onto adefined area of a substrate surface for specific binding to a samplemolecule, e.g., mRNA transcripts.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to an amylase and/or a glucoamylase of theinvention. These antibodies can be used to isolate, identify or quantifythe amylases and/or glucoamylases of the invention or relatedpolypeptides. These antibodies can be used to isolate other polypeptideswithin the scope the invention or other related amylases and/or aglucoamylase. The antibodies can be designed to bind to an active siteof an amylase and/or a glucoamylase. Thus, the invention providesmethods of inhibiting amylases and/or a glucoamylases using theantibodies of the invention.

The invention provides antibodies that comprise a peptide or polypeptidederived from, modeled after or substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof,capable of specifically binding an antigen or epitope, see, e.g.Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press,N.Y. (1993); Wilson (1994) J. Immunol. Methods 175:267-273; Yarmush(1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includesantigen-binding portions, i.e., “antigen binding sites,” (e.g.,fragments, subsequences, complementarity determining regions (CDRs))that retain capacity to bind antigen, including (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR). Singlechain antibodies are also included by reference in the term “antibody.”

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N Y (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

Polypeptides or peptides can be used to generate antibodies which bindspecifically to the polypeptides, e.g., the amylases and/or aglucoamylases of the invention. The resulting antibodies may be used inimmunoaffinity chromatography procedures to isolate or purify thepolypeptide or to determine whether the polypeptide is present in abiological sample. In such procedures, a protein preparation, such as anextract, or a biological sample is contacted with an antibody capable ofspecifically binding to one of the polypeptides of the invention.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of the invention.After a wash to remove non-specifically bound proteins, the specificallybound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays, and Western Blots.

Polyclonal antibodies generated against the polypeptides of theinvention can be obtained by direct injection of the polypeptides intoan animal or by administering the polypeptides to a non-human animal.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique, the trioma technique, thehuman B-cell hybridoma technique, and the EBV-hybridoma technique (see,e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (see,e.g., U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies to the polypeptides of the invention. Alternatively,transgenic mice may be used to express humanized antibodies to thesepolypeptides or fragments thereof.

Antibodies generated against the polypeptides of the invention may beused in screening for similar polypeptides (e.g., amylases and/or aglucoamylase) from other organisms and samples. In such techniques,polypeptides from the organism are contacted with the antibody and thosepolypeptides which specifically bind the antibody are detected. Any ofthe procedures described above may be used to detect antibody binding.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., amylases and/or a glucoamylase)and/or antibodies of the invention. The kits also can containinstructional material teaching the methodologies and industrial uses ofthe invention, as described herein.

Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a new or modified amylase and/or a glucoamylaseactivity, by modifying the genetic composition of the cell. The geneticcomposition can be modified by addition to the cell of a nucleic acid ofthe invention. To detect the new phenotype, at least one metabolicparameter of a modified cell is monitored in the cell in a “real time”or “on-line” time frame. In one aspect, a plurality of cells, such as acell culture, is monitored in “real time” or “on-line.” In one aspect, aplurality of metabolic parameters is monitored in “real time” or“on-line.” Metabolic parameters can be monitored using the amylasesand/or glucoamylases of the invention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., anamylase and/or a glucoamylase message) or generating new (e.g., amylaseand/or a glucoamylase) transcripts in a cell. This increased ordecreased expression can be traced by testing for the presence of anamylase and/or a glucoamylase of the invention or by amylase and/or aglucoamylase activity assays. mRNA transcripts, or messages, also can bedetected and quantified by any method known in the art, including, e.g.,Northern blots, quantitative amplification reactions, hybridization toarrays, and the like. Quantitative amplification reactions include,e.g., quantitative PCR, including, e.g., quantitative reversetranscription polymerase chain reaction, or RT-PCR; quantitative realtime RT-PCR, or “real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001)Br. J. Haematol. 114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., anamylase and/or a glucoamylase) or generating new polypeptides in a cell.This increased or decreased expression can be traced by determining theamount of amylase and/or a glucoamylase present or by amylase and/or aglucoamylase activity assays. Polypeptides, peptides and amino acidsalso can be detected and quantified by any method known in the art,including, e.g., nuclear magnetic resonance (NMR), spectrophotometry,radiography (protein radiolabeling), electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, variousimmunological methods, e.g. immunoprecipitation, immunodiffusion,immuno-electrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immuno-fluorescent assays, gelelectrophoresis (e.g., SDS-PAGE), staining with antibodies, fluorescentactivated cell sorter (FACS), pyrolysis mass spectrometry,Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, andLC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, andthe like. Novel bioactivities can also be screened using methods, orvariations thereof, described in U.S. Pat. No. 6,057,103. Furthermore,as discussed below in detail, one or more, or, all the polypeptides of acell can be measured using a protein array.

INDUSTRIAL APPLICATIONS

The invention provides many industrial uses and medical applications forthe amylases, glucoamylase, and glucosidases and other polypeptides(e.g., antibodies) of the invention. For example, the invention providesenzymes and methods for liquefying polysaccharide, e.g., starch. Manyamylases and/or a glucoamylases and glucosidases used in processes forconverting liquefied polysaccharide, e.g., starch, to glucose are unableto hydrolyze alpha (1,6) linkages, and this deficiency leavesapproximately 5% of the sugar as pannose and isomaltose. However, in oneaspect, enzymes of the invention can convert polysaccharide, e.g.,starch, to glucose to maximize glucose production, including convertingliquefied polysaccharide, e.g., starch, to glucose. In one aspect, theinvention provides enzymes and methods for hydrolyzing 1,4-alpha and/or1,6-alpha linkages (e.g., hydrolyzing starches) and hydrolyzing pannoseand isomaltase. The glucosidases of the invention can be used in avariety of industrial processes, including biomass conversion to fuels(e.g., biofuels, such as bioethanol, biopropanol, biobutanol, or abiodiesel) and the like), e.g., including their use in the initialstages (liquefaction) of polysaccharide, e.g., starch, processing, inwet corn milling, in alcohol production, in the textile industry forpolysaccharide, e.g., starch, desizing, in baking applications, in thebeverage industry, in oilfields in drilling processes; in inking ofrecycled paper; and in animal feed. Thus, the invention also provides afuel, e.g., a biofuel (such as a bioethanol, biopropanol, biobutanol, ora biodiesel), comprising a polypeptide of the invention.

Detergent Compositions

The invention provides detergent compositions comprising one or morepolypeptides of the invention, for example, amylases and/orglucoamylases of the invention, such as alpha amylases, etc., andmethods of making and using these compositions. The inventionincorporates all methods of making and using detergent compositions,see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561; 6,365,561; 6,380,147.The detergent compositions can be a one and two part aqueouscomposition, a non-aqueous liquid composition, a cast solid, a granularform, a particulate form, a compressed tablet, a gel and/or a paste anda slurry form. The invention also provides methods capable of a rapidremoval of gross food soils, films of food residue and other minor foodcompositions using these detergent compositions. Amylases and/orglucoamylases of the invention can facilitate the removal ofpolysaccharide-comprising, e.g., starchy, stains by means of catalytichydrolysis of a polysaccharide and/or oligosaccharide, e.g., starch.Amylases and/or glucoamylases of the invention can be used indishwashing detergents in textile laundering detergents.

The actual active enzyme content depends upon the method of manufactureof a detergent composition and is not critical, assuming the detergentsolution has the desired enzymatic activity. In one aspect, the amountof amylase and/or glucoamylase present in the final solution ranges fromabout 0.001 mg to 0.5 mg per gram of the detergent composition. Theparticular enzyme chosen for use in the process and products of thisinvention depends upon the conditions of final utility, including thephysical product form, use pH, use temperature, and soil types to bedegraded or altered. The enzyme can be chosen to provide optimumactivity and stability for any given set of utility conditions. In oneaspect, the polypeptides of the present invention are active in the pHranges of from about 4 to about 12 and in the temperature range of fromabout 20° C. to about 95° C. The detergents of the invention cancomprise cationic, semi-polar nonionic or zwitterionic surfactants; or,mixtures thereof.

Amylases and/or glucoamylases of the present invention can be formulatedinto powdered and liquid detergents having pH between 4.0 and 12.0 atlevels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight.These detergent compositions can also include other enzymes such asknown proteases, cellulases, lipases or endoglycosidases, as well asbuilders and stabilizers. The addition of amylases and/or glucoamylasesof the invention to conventional cleaning compositions does not createany special use limitation. In other words, any temperature and pHsuitable for the detergent is also suitable for the present compositionsas long as the pH is within the above range, and the temperature isbelow the described enzyme's denaturing temperature. In addition, thepolypeptides of the invention can be used in a cleaning compositionwithout detergents, again either alone or in combination with buildersand stabilizers.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. In one aspect, a polypeptide ofthe invention (e.g., an alkaline-active amylase and/or glucoamylase) isused in a detergent, i.e., as a detergent additive. The detergentcomposition of the invention may, for example, be formulated as a handor machine laundry detergent composition comprising a polypeptide of theinvention. Detergent compositions of the invention include laundry anddishwashing (e.g., autodishwashing) solutions and application. A laundryadditive suitable for pre-treatment of stained fabrics can comprise apolypeptide of the invention. A fabric softener composition can comprisea polypeptide of the invention. Alternatively, a polypeptide of theinvention can be formulated as a detergent composition for use ingeneral household hard surface cleaning operations. In alternativeaspects, detergent additives and detergent compositions of the inventionmay comprise one or more other enzymes such as a protease, a lipase, acutinase, another amylase and/or glucoamylase, a carbohydrase, acellulase, a pectinase, a mannanase, an arabinase, a galactanase, axylanase, an oxidase, e.g., a lactase, and/or a peroxidase. Theproperties of the enzyme(s) of the invention are chosen to be compatiblewith the selected detergent (i.e. pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.) and the enzyme(s) ispresent in effective amounts. In one aspect, amylase and/or glucoamylaseenzymes of the invention are used to remove malodorous materials fromfabrics. Various detergent compositions and methods for making them thatcan be used in practicing the invention are described in, e.g., U.S.Pat. Nos. 6,333,301; 6,329,333; 6,326,341; 6,297,038; 6,309,871;6,204,232; 6,197,070; 5,856,164.

Treating Fabrics

The invention provides methods of treating fabrics using one or morepolypeptides of the invention. The polypeptides of the invention can beused in any fabric-treating method, which are well known in the art,see, e.g., U.S. Pat. No. 6,077,316. For example, in one aspect, the feeland appearance of a fabric is improved by a method comprising contactingthe fabric with an amylase and/or a glucoamylase of the invention in asolution. In one aspect, the fabric is treated with the solution underpressure.

In one aspect, the enzymes of the invention are applied during or afterthe weaving of textiles, or during the desizing stage, or one or moreadditional fabric processing steps. During the weaving of textiles, thethreads are exposed to considerable mechanical strain. Prior to weavingon mechanical looms, warp yarns are often coated with sizing starch orstarch derivatives in order to increase their tensile strength and toprevent breaking. The enzymes of the invention can be applied to removethese sizing starch or starch derivatives. After the textiles have beenwoven, a fabric can proceed to a desizing stage. This can be followed byone or more additional fabric processing steps. Desizing is the act ofremoving size from textiles. After weaving, the size coating must beremoved before further processing the fabric in order to ensure ahomogeneous and wash-proof result. The invention provides a method ofdesizing comprising enzymatic hydrolysis of the size by the action of anenzyme of the invention.

The enzymes of the invention can be used to desize fabrics, includingcotton-containing fabrics, as detergent additives, e.g., in aqueouscompositions. The invention provides methods for producing a stonewashedlook on indigo-dyed denim fabric and garments. For the manufacture ofclothes, the fabric can be cut and sewn into clothes or garments, whichis afterwards finished. In particular, for the manufacture of denimjeans, different enzymatic finishing methods have been developed. Thefinishing of denim garment normally is initiated with an enzymaticdesizing step, during which garments are subjected to the action ofamylolytic enzymes in order to provide softness to the fabric and makethe cotton more accessible to the subsequent enzymatic finishing steps.The invention provides methods of finishing denim garments (e.g., a“bio-stoning process”), enzymatic desizing and providing softness tofabrics using the amylases and/or glucoamylases of the invention. Theinvention provides methods for quickly softening denim garments in adesizing and/or finishing process.

Foods and Food Processing: Liquefaction of Polysaccharides, e.g., Starch

The enzymes of the invention have numerous applications in foodprocessing industry. The amylases and/or glucoamylases of the inventionare used in starch to fructose processing. In alternative aspect,processes of the invention comprise polysaccharide, e.g., starch, tofructose processing comprising four steps: liquefaction of granularstarch, saccharification of the liquefied polysaccharide, e.g., starch,into dextrose, purification, and isomerization to fructose; and one,several or all of these steps can comprise use of one or more enzymes ofthe invention. Enzymes of the invention can be used in ethanolproduction from a biomass, e.g., a corn or a grass, by wet or by drymilling; for example, FIG. 12 illustrates how enzymes of the inventioncan be used in ethanol production from corn by dry milling, includingtheir use in both “conventional processes” and “simultaneousliquefaction saccharification and fermentation processes” The inventionprovides methods of polysaccharide, e.g., starch, liquefaction using theenzymes of the invention. Concentrated suspensions of starch polymergranules are converted into a solution of soluble shorter chain lengthdextrins of low viscosity. This step is useful for convenient handlingwith standard equipment and for efficient conversion to glucose or 10³other sugars. In one aspect, the granular starch is liquefied bygelatinizing the granules by raising the temperature of the granularstarch to over about 72° C. The heating process instantaneously disruptsthe insoluble starch granules to produce a water soluble starchsolution. The solubilized starch solution can then be liquefied by anamylase and/or a glucoamylase of the invention. Thus, the inventionprovides enzymatic starch liquefaction processes using an amylase and/ora glucoamylase of the invention.

FIG. 7, FIG. 8 and FIG. 9 illustrate alternative exemplary starchprocesses, including starch liquefaction processes, of the inventionusing at least one enzyme of the invention. For example, FIG. 7illustrates an exemplary starch liquefaction process of the inventioncomprising treating a starch slurry (e.g., having about 30% to 35%solids) with steam for primary liquefaction (e.g., at about 105° C. forabout 5 minutes), input into a flash tank, followed by secondaryliquefaction (e.g., at about 90° C. to 95° C. for about 90 minutes),each or one of these steps involving use of an enzyme of the invention.FIG. 8 illustrates another exemplary starch liquefaction process of theinvention comprising treating a starch slurry at about between pH 4 topH 5, e.g., pH 4.5, adjusting the pH, calcium addition, liquefaction atabout pH 5 to pH 6, e.g., pH 5.4, at about 95° C. using an amylase(e.g., alpha amylase) and/or glucoamylase of the invention, followed byanother pH and temperature adjustment for saccharification at aboutbetween pH 4 to pH 5, e.g., pH 4.5, at a temperature of between about60° C. to 65° C. using an amylase (e.g., alpha amylase) and/orglucoamylase of the invention. FIG. 9 illustrates another exemplarystarch process of the invention comprising treating a starch slurry atabout between pH 4 to pH 5, e.g., pH 4.5, (optional adjusting pH,calcium addition), combined liquefaction-saccharification using an alphaamylase and/or a glucoamylase of the invention at about between pH 4 topH 5, e.g., pH 4.5, at a temperature of greater than about 90° C., or,greater than about 95° C., followed by another pH and temperatureadjustment for saccharification at about between pH 4 to pH 5, e.g., pH4.5, at a temperature of between about 60° C. to 65° C. using aglucoamylase of the invention. In one aspect, the combinedliquefaction-saccharification of the invention is a “one-pot” process.In one aspect, the entire process is a “one-pot” process. Any one ofthese processes, and any one of these steps, can also comprise, or canfurther comprise, another enzyme of the invention (e.g., a glucosidasesuch as an alpha-1,6-glucosidase, a maltase, etc.), or another enzymesuch as a pullulanase or an isomerase.

An exemplary enzymatic liquefaction process involves adjusting the pH ofa granular starch slurry to between 6.0 and 6.5 and the addition ofcalcium hydroxide, sodium hydroxide or sodium carbonate. In one aspect,calcium hydroxide is added. This provides calcium ions to stabilize theglucoamylase of the invention against inactivation. In one aspect, uponaddition of amylase, the suspension is pumped through a steam jet toinstantaneously raise the temperature to between 80°-115° C. In oneaspect, the starch is immediately gelatinized and, due to the presenceof amylase, depolymerized through random hydrolysis ofalpha-1,4-glycosidic bonds by amylase to a fluid mass. The fluid masscan be easily pumped.

The invention provides various enzymatic polysaccharide, oligosaccharideand/or starch liquefaction processes using an amylase and/or aglucoamylase of the invention. In one aspect of the liquefaction processof the invention, an amylase is added to the polysaccharide,oligosaccharide and/or starch suspension and the suspension is held at atemperature of between about 80°-100° C. to partially hydrolyze thepolysaccharide, oligosaccharide and/or starch granules. In one aspect,the partially hydrolyzed polysaccharide, oligosaccharide and/or starchsuspension is pumped through a jet at temperatures in excess of about105° C. to thoroughly gelatinize any remaining granular structure. Inone aspect, after cooling the gelatinized starch, a second addition ofamylase and/or glucoamylase is made to further hydrolyze thepolysaccharide, oligosaccharide and/or starch.

The invention provides enzymes and processes for hydrolyzing liquid(liquefied) and granular polysaccharide, oligosaccharide and/or starch.Such starch can be derived from any source, e.g., corn, wheat, milo,sorghum, rye or bulgher. The invention applies to any grain starchsource which is useful in liquefaction, e.g., any other grain orvegetable source known to produce starch suitable for liquefaction. Themethods of the invention comprise liquefying starch from any naturalmaterial, such as rice, germinated rice, corn, barley, milo, wheat,legumes and sweet potato. The liquefying process can substantiallyhydrolyze the starch to produce a syrup. The temperature range of theliquefaction can be any liquefaction temperature which is known to beeffective in liquefying starch. For example, the temperature of thepolysaccharide, oligosaccharide and/or starch can be between about 80°C. to about 115° C., between about 100° C. to about 110° C., and fromabout 105° C. to about 108° C. In alternative aspects, the amylaseand/or glucoamylase used in these methods is active at thesetemperatures, e.g., active at temperatures in a range of between about80° C. to about 115° C., between about 100° C. to about 110° C., andfrom about 105° C. to about 108° C.

The invention provides methods for liquefaction saccharification asillustrated in FIG. 5. In one aspect, amylases (such as alpha amylases)and/or glucoamylases of the invention are used in the illustratedliquefaction step (some current industrial methods use B. licheniformisalpha-amylase). In one aspect, the process takes place at about pH 6.0at a temperature anywhere in the range of between about 95° C. to 105°C., for a length of time anywhere between about 0.5 and 5 hours, e.g.,60, 90 or 120 minutes. In one aspect, in a corn steep process, prior toliquefaction cellulases, proteases and/or protein thioreductases areadded.

In one aspect of a liquefaction process of the invention, an amylaseand/or a glucoamylase of the invention that has activity at about pH 4.5(or, anywhere between about pH 5 and pH 5), that may or may not be Ca²⁺dependent is added. Eliminating the addition of salts in the front endof the process eliminates the need to remove them at the back end of theprocess. In one aspect of a liquefaction process of the invention, anamylase that is more active is used. This can allow one to decrease theamount of enzyme needed. In one aspect, liquefaction andsaccharification are done in the same pot, as a “one-pot process,” forexample, under conditions comprising about 90° C. to 95° C. (or,anywhere between about 80° C. to 105° C.), as about a 3 hour process(or, as a process lasting between about 1 and 5 hours). In this aspect,the enzyme load can be cut in half again.

In one aspect of a saccharification process of the invention, an amylaseand/or glucoamylase of the invention is used. In one aspect, amylasesand/or glucoamylases of the invention are used in a saccharificationstep (in addition to or in place of an A. niger glucoamylase). In oneaspect, the process takes place at about pH 4.5, in a temperature rangeof between about 60° C. to 62° C. (or, anywhere in the range of betweenabout 50° C. to 72° C., or, between about 40° C. to 80° C.) as a processlasting between about 12 and 96 or more hours. In one aspect of asaccharification process of the invention, a glucoamylase of theinvention is used to give a higher level of dextrose in the syrup. Inone aspect, other enzymes are added, e.g., pullulanases to increase theamount of glucose.

In one aspect, one, some or all of the enzymes used in processes of theinvention (including the enzymes of the invention) are immobilized,e.g., immobilized on any surface, e.g., a flat surface or an enzymecolumn, e.g., immobilized on an array, a bead, fiber, pore, capillaryand the like. In one aspect, by being immobilized, they can be reused.

In one aspect, the invention provides “enzyme cocktails” using at leastone enzyme of the invention. In one aspect, “enzyme cocktails” are usedin the processes of the invention, e.g., including the liquefactionsaccharification methods as illustrated in FIG. 5. For example, in oneaspect, cell wall degrading enzymes (CWDE) are used, e.g., for textile,pulp and paper, and laundry processes of the invention, including, e.g.,combinations of cellulases, hemicellulases, xylanase, galactomannanases,gluco-mannanases, arabinofuranosidases, and others. In one aspect,“enzyme cocktails” used in the processes of the invention forbio-bleaching (e.g., pulp and paper, laundry processes), includecombinations of laccases, peroxidases, oxidases and the like. In oneaspect, cell wall degrading enzymes are combined with bio-bleachingenzymes and enzymes of the invention to degrade plant cell walls torelease color agents.

One exemplary enzyme cocktail of the invention comprising at least oneamylase and/or glucoamylase of the invention can hydrolyze approximatelygreat than 95% of the starch in milled corn into fermentable sugars inno more than 60 hours at about 30 to 40° C. and at about pH 3.5 to pH5.5 in the presence of yeast. In one aspect, the total quantity ofenzyme protein required is not greater than 50 grams/ton of corn (0.005%w/w).

Enzymes of the invention can be used to treat dairy products; and theinvention provides dairy products comprising a polypeptide of theinvention, wherein optionally the dairy product comprises a milk, an icecream, a cheese or a yogurt.

Enzymes of the invention can be used in simultaneous liquefactionsaccharification and fermentation (SLSF) processes; and advantages tousing enzymes of this invention can include:

-   -   Lower energy costs due to elimination of high temperature step;    -   Improved yield due to more rapid fermentation;    -   Reduced risk of bacterial contamination;    -   yeast produces ethanol earlier than in conventional process;    -   Reduced storage and handling of bulk yeast.

Properties of enzymes of the invention used in these processes of theinvention can include: raw polysaccharide, oligosaccharide and/or starchhydrolyzing activity; temperature and pH activity profiles compatiblewith process conditions; high activity on non-gelatinizedpolysaccharide, oligosaccharide and/or starch; and/or active on‘resistant’ fraction of raw starch.

In alternative aspects, enzymes of the invention have amylase and/orglucoamylase activity and can hydrolyze (partially or completely)polysaccharide, oligosaccharide and/or starch granules having complexmulti-level structures, including amorphous and crystalline regionsand/or branched and linear chains, e.g., can hydrolyze (partially orcompletely) raw starch, including: RS I—physically inaccessible starch;RS II—resistant starch granules as in raw potato and banana; RSIII—retrograded as in cooked potato.

Processes to Produce High MW Dextrose Syrups

The invention provides processes to produce high MW dextrose syrupsusing enzymes of the invention, including methods for producingoligosaccharides having a MW tightly groups at about 20,000 MW. In oneaspect, amylases and/or glucoamylases of the invention can be used toliquefy a polysaccharide-comprising, oligosaccharide-comprising and/orstarch starch-comprising composition, e.g., a corn starch, to produce anoligosaccharide pattern that is tightly grouped at about 20,000 MW(Bacillus amylases will produce syrups containing much higher MWfragments, and high MW oligosaccharides are not fully converted toglucose by glucoamylases, e.g., Aspergillus glucoamylases, duringsaccharification).

In one aspect, using the amylases and/or glucoamylases of the inventionto catalyze the hydrolysis of a starch-comprising composition, e.g., acorn starch, the approximately 20,000 MW fragments are produced. Theseapproximately 20,000 MW fragments can be rapidly and fully converted toglucose. Thus, in one aspect, saccharified syrups resulting fromBacillus amylase liquefaction contain less dextrose than saccharifiedsyrups from liquefaction using amylases and/or glucoamylases of theinvention.

Processes to Produce Homogenous Maltodextrins

The invention provides processes to produce homogenous maltodextrinsusing enzymes of the invention. The homogenous maltodextrins produced bythe methods of the invention can be used in a wide variety of food, drugand coating applications. In one aspect, amylases and/or glucoamylasesof the invention can be used to generate an extremely uniformmaltodextrin composition (conventional manufacturing processes usingeither acid or enzymatic hydrolysis of starch result in a broad,typically bimodal MW distribution of oligosaccharides). The homogenousmaltodextrins produced by the methods of the invention have a homogenousMW distribution and can be used in a variety of maltodextrin-comprisingproducts, resulting in lower viscosity, clear (no haze) solutions,better coating properties, better film-forming properties, and the like.

In one aspect, amylases and/or glucoamylases of the invention are usedto liquefy corn starch to produce a uniform maltodextrin-comprisingcomposition. In one aspect, the liquefaction is conducted at a pH ofbetween about pH 4.5 to about pH 6.5, e.g., pH 5.0 or 5.5, attemperatures up to about 105° C. The uniform maltodextrin compositioncan be produced at DE's ranging from about 5 to as high as about 20. Thesyrups produced by these amylases and/or glucoamylases of the inventioncan be filtered, treated with charcoal and/or spray-dried to yield themaltodextrin-comprising product.

In one aspect, one or more other enzymes are used in conjunction with acomposition comprising enzymes of the invention for use in starch tofructose processing, liquefaction of granular starch, and processes toproduce homogenous maltodextrins or high MW dextrose syrups, e.g.,including other amylases, beta-galactosidases, catalases, laccases,cellulases, endoglycosidases, endo-beta-1,4-laccases, amyloglucosidases,other glucosidases, glucose isomerases, glycosyltransferases, lipases,phospholipases, lipooxygenases, beta-laccases,endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,glucoamylases, pectinases, reductases, oxidases, decarboxylases,phenoloxidases, ligninases, pullulanases, arabinanases, hemi cellulases,mannanases, xylolaccases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases,polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases,transglutaminases, pectin methylesterases, cellobiohydrolases and/ortransglutaminases.

Enzymatic Dry Milling Processes

The invention provides enzymatic dry milling processes using an amylaseand/or a glucoamylase of the invention; exemplary processes areillustrated in FIG. 12. In dry milling, whole grain is ground andcombined with water. The germ is optionally removed by flotationseparation or equivalent techniques. The resulting mixture, whichcontains polysaccharide, e.g., starch, fiber, protein and othercomponents of the grain, is liquefied using amylase. In one aspect,enzymatic liquefaction is done at lower temperatures than thepolysaccharide, e.g., starch, liquefaction processes discussed above. Inone aspect, after gelatinization the polysaccharide, e.g., starch,solution is held at an elevated temperature in the presence of amylaseuntil a DE of 10-20 is achieved. In one aspect, this is a period ofabout 1-3 hours. Dextrose equivalent (DE) is the industry standard formeasuring the concentration of total reducing sugars, calculated asD-glucose on a dry weight basis. Unhydrolyzed granular starch has a DEof virtually zero, whereas the DE of D-glucose is defined as 100.

Enzymes of the invention can be used in biomass wet or dry millingprocesses. For example, in one aspect of an exemplary dry millingprocess whole grain is ground and combined with water and treated withan enzyme of the invention. The germ can be removed by flotationseparation or equivalent techniques. In one aspect, the resultingmixture, which contains polysaccharide, e.g., starch, fiber, protein andother components of the grain, is liquefied using a glucoamylase and/oran amylase of the invention (e.g., an alpha-amylase). In one aspect,this enzymatic liquefaction is at a relatively lower temperature whenusing the dry milling process; however, low temperature liquefaction isbelieved to be less efficient than high temperature liquefaction inconverting polysaccharide, e.g., starch, to soluble dextrins. Thus, inone aspect, the invention provides a further step wherein aftergelatinization the polysaccharide, e.g., starch, solution is held at anelevated temperature in the presence of a glucoamylase and/or an amylaseof the invention (e.g., an alpha-amylase), which in one aspect is untila DE of about 10 to 20 is achieved, usually a period of about 1 to 3hours (dextrose equivalent (DE) is the industry standard for measuringthe concentration of total reducing sugars, calculated as D-glucose on adry weight basis; unhydrolyzed granular starch has a DE of virtuallyzero, whereas the DE of D-glucose is defined as 100).

In alternative aspects, use of amylases and/or glucoamylases of theinvention in dry mill ethanol processes can provide operationaladvantages, for example: rapid reduction in viscosity of slurried cornflour, making an increase in dissolved solids and throughput possiblewithout additional capital investment; superior thermal stability tobest competitor, which eliminates split dosing; some amylases and/orglucoamylases of the invention are thermostable enzymes—and thiseliminates the need to dose before jet cooking and after; lowerviscosities are obtained at higher process temperatures, and providesimproved microbial control in slurry tank (process is run at highertemperature, so unwanted microbes are killed); lower liquefaction pH,which eliminates need for pH adjustment, decreases scale formation(calcium oxalate precipitate forms on hardware, etc.; if liquefactiondone at low pH, there is a higher potential for scale formation) andreduces byproduct formation.

In summary, in alternative aspects amylases and/or glucoamylases of theinvention can be thermostable enzymes that can meet key industry needs,for example, under certain conditions, rapidly reduces viscosity of highdry solids corn flour slurry, can be thermostable (optimum temperature95° C.), can be calcium independent, can be active under low pH optimum,and can tolerate up to 30% recycled backset. In one aspect, therecommended dose is in the range of between about 0.4 to 0.6 kg/MTstarch.

Enzymatic Wet Milling Processes

The invention provides wet milling processes, e.g., corn wet milling,using an enzyme, e.g., a glucoamylase and/or an amylase, of theinvention. Corn wet milling is a process which produces corn oil, glutenmeal, gluten feed and polysaccharide, e.g., starch. Thus, the inventionprovides methods of making corn oil, gluten meal, gluten feed andpolysaccharide, e.g., starch, using an enzyme of the invention. In oneaspect, an alkaline-amylase and/or alkaline-glucoamylase of theinvention is used in the liquefaction of polysaccharide, e.g., starch.In one aspect, an amylase and/or glucoamylase of the invention is usedin saccharification to produce glucose. An exemplary corn wet millingprocess of the invention (using at least one enzyme of the invention) isillustrated in FIG. 6. FIG. 6 illustrates an exemplary corn oil processof the invention comprising steeping, de-germing, de-fibering and glutenseparation, followed by liquefaction using an enzyme of the invention(e.g., an alpha amylase), and saccharification using an enzyme of theinvention (e.g., glucoamylase).

In one aspect, corn (a kernel that consists of a outer seed coat(fiber), polysaccharide, e.g., starch, a combination of starch andglucose and the inner germ), is subjected to a four step process, whichresults in the production of starch. In one aspect, the corn is steeped,de-germed, de-fibered, and the gluten is separated. In a steepingprocess the solubles are taken out. The product remaining after removalof the solubles is de-germed, resulting in production of corn oil andproduction of an oil cake, which is added to the solubles from thesteeping step. The remaining product is de-fibered and the fiber solidsare added to the oil cake/solubles mixture. This mixture of fibersolids, oil cake and solubles forms a gluten feed. After de-fibering,the remaining product is subjected to gluten separation. This separationresults in a gluten meal and starch. The starch is then subjected toliquefaction and saccharification using polypeptides of the invention toproduce glucose.

FIG. 6 illustrates an exemplary corn wet milling process of theinvention (using at least one enzyme of the invention). FIG. 7, FIG. 8and FIG. 9 illustrate alternative exemplary starch processing methods(e.g., industrial processes), including starch liquefaction processes,of the invention (using at least one enzyme of the invention).

Enzymes of the invention can be used in biomass wet dry millingprocesses; e.g., a corn wet milling, to produces a vegetable (e.g., acorn) oil, a gluten meal, a gluten feed and/or a starch. In one aspect,an alkaline-amylase of the invention is used in the liquefaction ofstarch and a glucoamylases (which also can be an enzyme of theinvention) is used in saccharification, producing glucose. In oneaspect, the biomass (e.g., a corn kernel, which consists of an outerseed coat (fiber), starch, a combination of starch and glucose and theinner germ) is subjected to a four step process to produce starch. Thebiomass (e.g., a corn) is steeped, de-germed, de-fibered, and finallythe gluten is separated. In the steeping process, the solubles are takenout. The product remaining after removal of the solubles is de-germed,resulting in production of a vegetable oil (e.g., a corn oil) andproduction of an oil cake, which is added to the solubles from thesteeping step. The remaining product is de-fibered and the fiber solidsare added to the oil cake/solubles mixture. This mixture of fibersolids, oil cake and solubles forms a gluten feed. After de-fibering,the remaining product is subjected to gluten separation. This separationresults in a gluten meal and starch. In one aspect, the starch thensubjected to liquefaction and saccharification (e.g., using enzymes ofthe invention) to produce glucose.

The invention also provides a high yield process for producing highquality corn fiber gum by treatment of corn fiber with an enzyme of theinvention followed by hydrogen peroxide treatment to obtain an extractof milled corn fiber. See, e.g., U.S. Pat. No. 6,147,206.

In one aspect, one or more other enzymes are used in conjunction with acomposition comprising enzymes of the invention for use with these dryor wet milling processes, e.g., including other amylases,beta-galactosidases, catalases, laccases, cellulases, endoglycosidases,endo-beta-1,4-laccases, amyloglucosidases, other glucosidases, glucoseisomerases, glycosyltransferases, lipases, phospholipases,lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases, cutinases,peroxidases, amylases, glucoamylases, pectinases, reductases, oxidases,decarboxylases, phenoloxidases, ligninases, pullulanases, arabinanases,hemi cellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, proteases, peptidases,proteinases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases.

Anti-Staling Processes

The invention provides anti-staling processes (e.g., of baked productssuch as bread) using an amylase and/or a glucoamylase of the invention.The invention provides methods to slow the increase of the firmness ofthe crumb (of the baked product) and a decrease of the elasticity of thecrumb using an amylase and/or a glucoamylase of the invention. Stalingof baked products (such as bread) is more serious as time passes betweenthe moment of preparation of the bread product and the moment ofconsumption. The term staling is used to describe changes undesirable tothe consumer in the properties of the bread product after leaving theoven, such as an increase of the firmness of the crumb, a decrease ofthe elasticity of the crumb, and changes in the crust, which becomestough and leathery. The firmness of the bread crumb increases furtherduring storage up to a level, which is considered as negative. Amylasesand/or glucoamylases of the invention are used to retard staling of thebread as described e.g., in U.S. Pat. Nos. 6,197,352; 2,615,810;3,026,205; Silberstein (1964) Baker's Digest 38:66-72.

In one aspect, an enzyme of the invention is used to retard the stalingof baked products while not hydrolyzing starch into the brancheddextrins. Branched dextrins are formed by cleaving off the branchedchains of the dextrins generated by alpha-amylase hydrolysis whichcannot be degraded further by the alpha-amylase. This can produce agummy crumb in the resulting bread. Accordingly, the invention providesa process for retarding the staling of baked products (e.g., leavenedbaked products) comprising adding an enzyme of the invention comprisingexoamylase activity to a flour or a dough used for producing a bakedproduct. Exoamylases and/or glucoamylases of the invention can haveglucoamylase, β-amylase (which releases maltose in thebeta-configuration) and/or maltogenic amylase activity.

The invention also provides a process for preparing a dough or a bakedproduct prepared from the dough which comprises adding an amylase and/ora glucoamylase of the invention to the dough in an amount which iseffective to retard the staling of the bread. The invention alsoprovides a dough comprising said amylase and a premix comprising flourtogether with said amylase. Finally, the invention provides an enzymaticbaking additive, which contains said amylase.

Animal Feeds and Additives

The invention provides feeds, foods, food additives, feed additives,nutritional supplements and/or dietary supplements comprising apolypeptide of this invention for humans and animals; and the inventionprovides methods for treating humans and animals using feeds, foods,food additives, feed additives, nutritional supplements and/or dietarysupplements comprising a polypeptide of this invention; and/or using aglucoamylase and/or amylase enzyme of the invention. The inventionprovides human and/or animal feeds, foods, food additives, feedadditives, nutritional supplements and/or dietary supplements comprisingamylases and/or glucoamylases of the invention. In one aspect, treatinghuman and/or animal feeds, foods, additives, food additives, feedadditives, nutritional supplements and/or dietary supplements usingamylase and/or glucoamylases enzymes of the invention can help in theavailability of polysaccharide, e.g., starch, in the human and/or animalfeed, food, additive, food additive, feed additive, nutritionalsupplement and/or dietary supplement. This can result in release ofreadily digestible and easily absorbed sugars.

Use of a glucoamylase and/or an amylase of the invention can increasethe digestive capacity of animals and birds. Use of an amylase and/or aglucoamylase of the invention can ensure availability of an adequatenutrient supply for better growth and performance. In one aspect, theenzymes of the invention can be added as feed additives, or in feeds,foods, additives, food additives, feed additives, nutritionalsupplements and/or dietary supplements, for animals. In another aspect,the feeds, foods, additives, food additives, feed additives, nutritionalsupplements and/or dietary supplements, e.g., animal feed, can betreated with an amylase and/or a glucoamylase of the invention prior toanimal consumption. In another aspect, an amylase and/or a glucoamylaseof the invention may be supplied by expressing the enzymes directly intransgenic feed crops (as, e.g., transgenic plants, seeds and the like),such as corn. As discussed above, the invention provides transgenicplants, plant parts and plant cells comprising a nucleic acid sequenceencoding a polypeptide of the invention. In one aspect, the nucleic acidis expressed such that an amylase and/or a glucoamylase of the inventionis produced in recoverable quantities. The amylase and/or glucoamylaseof the invention can be recovered from any plant or plant part.Alternatively, the plant or plant part containing the recombinantpolypeptide can be used as such for improving the quality of a food orfeed, feed additive, nutritional supplement and/or dietary supplement,and the like, e.g., improving nutritional value, palatability, andrheological properties, or to destroy an antinutritive factor.

Paper or Pulp Treatment

The enzymes of the invention can be in paper or pulp treatment or paperdeinking. For example, in one aspect, the invention provides a papertreatment process using amylases and/or glucoamylases of the invention.In one aspect, the enzymes of the invention can be used to modifypolysaccharide, e.g., starch, in the paper thereby converting it into aliquefied form. In another aspect, paper components of recycledphotocopied paper during chemical and enzymatic deinking processes. Inone aspect, amylases and/or glucoamylases of the invention can be usedin combination with cellulases. The paper can be treated by thefollowing three processes: 1) disintegration in the presence of anenzyme of the invention, 2) disintegration with a deinking chemical andan enzyme of the invention, and/or 3) disintegration after soaking withan enzyme of the invention. The recycled paper treated with amylase canhave a higher brightness due to removal of toner particles as comparedto the paper treated with just cellulase. While the invention is notlimited by any particular mechanism, the effect of an amylase and/or aglucoamylase of the invention may be due to its behavior assurface-active agents in pulp suspension.

The invention provides methods of treating paper and paper pulp usingone or more polypeptides of the invention. The polypeptides of theinvention can be used in any paper- or pulp-treating method, which arewell known in the art, see, e.g., U.S. Pat. Nos. 6,241,849; 6,066,233;5,582,681. For example, in one aspect, the invention provides a methodfor deinking and decolorizing a printed paper containing a dye,comprising pulping a printed paper to obtain a pulp slurry, anddislodging an ink from the pulp slurry in the presence of an enzyme ofthe invention (other enzymes can also be added). In another aspect, theinvention provides a method for enhancing the freeness of pulp, e.g.,pulp made from secondary fiber, by adding an enzymatic mixturecomprising an enzyme of the invention (can also include other enzymes,e.g., pectinase enzymes) to the pulp and treating under conditions tocause a reaction to produce an enzymatically treated pulp. The freenessof the enzymatically treated pulp is increased from the initial freenessof the secondary fiber pulp without a loss in brightness.

In one aspect, one or more other enzymes are used in conjunction with acomposition comprising enzymes of the invention for use with these pulpand/or paper treatment methods, e.g., including other amylases,beta-galactosidases, catalases, laccases, cellulases, endoglycosidases,endo-beta-1,4-laccases, amyloglucosidases, other glucosidases, glucoseisomerases, glycosyltransferases, lipases, phospholipases,lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases, cutinases,peroxidases, amylases, glucoamylases, pectinases, reductases, oxidases,decarboxylases, phenoloxidases, ligninases, pullulanases, arabinanases,hemi cellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, proteases, peptidases,proteinases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases.

Repulping: Treatment of Lignocellulosic Materials

The invention also provides a method for the treatment oflignocellulosic fibers, wherein the fibers are treated with apolypeptide of the invention, in an amount which is efficient forimproving the fiber properties. The amylases and/or glucoamylases of theinvention may also be used in the production of lignocellulosicmaterials such as pulp, paper and cardboard, from polysaccharide, e.g.,starch, reinforced waste paper and cardboard, especially where repulpingoccurs at pH above 7 and where amylases can facilitate thedisintegration of the waste material through degradation of thereinforcing polysaccharide, e.g., starch. The amylases and/orglucoamylases of the invention can be useful in a process for producinga papermaking pulp from starch-coated printed paper. The process may beperformed as described in, e.g., WO 95/14807.

An exemplary process comprises disintegrating the paper to produce apulp, treating with a polysaccharide-degrading, e.g., starch-degrading,enzyme before, during or after the disintegrating, and separating inkparticles from the pulp after disintegrating and enzyme treatment. Seealso U.S. Pat. No. 6,309,871 and other US patents cited herein. Thus,the invention includes a method for enzymatic deinking of recycled paperpulp, wherein the polypeptide is applied in an amount which is efficientfor effective deinking of the fiber surface.

Waste Treatment

The enzymes of the invention can be used in a variety of otherindustrial applications, e.g., in waste treatment. For example, in oneaspect, the invention provides a solid waste digestion process usingenzymes of the invention. The methods can comprise reducing the mass andvolume of substantially untreated solid waste. Solid waste can betreated with an enzymatic digestive process in the presence of anenzymatic solution (including an enzyme of the invention) at acontrolled temperature. This results in a reaction without appreciablebacterial fermentation from added microorganisms. The solid waste isconverted into a liquefied waste and any residual solid waste. Theresulting liquefied waste can be separated from said any residualsolidified waste. See e.g., U.S. Pat. No. 5,709,796.

In one aspect, one or more other enzymes are used in conjunction with acomposition comprising enzymes of the invention for use with these wastetreatment methods, e.g., including other amylases, beta-galactosidases,catalases, laccases, cellulases, endoglycosidases,endo-beta-1,4-laccases, amyloglucosidases, other glucosidases, glucoseisomerases, glycosyltransferases, lipases, phospholipases,lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases, cutinases,peroxidases, amylases, glucoamylases, pectinases, reductases, oxidases,decarboxylases, phenoloxidases, ligninases, pullulanases, arabinanases,hemi cellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, proteases, peptidases,proteinases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases.

Oral Care Products

The invention provides oral care product comprising an amylase and/or aglucoamylase of the invention. Exemplary oral care products includetoothpastes, dental creams, gels or tooth powders, odontics, mouthwashes, pre- or post brushing rinse formulations, chewing gums,lozenges, or candy. See, e.g., U.S. Pat. No. 6,264,925.

In one aspect, one or more other enzymes are used in conjunction with anoral care composition comprising enzymes of the invention, and for usewith these methods, e.g., including other amylases, beta-galactosidases,catalases, laccases, cellulases, endoglycosidases,endo-beta-1,4-laccases, amyloglucosidases, other glucosidases, glucoseisomerases, glycosyltransferases, lipases, phospholipases,lipooxygenases, beta-laccases, endo-beta-1,3(4)-laccases, cutinases,peroxidases, amylases, glucoamylases, pectinases, reductases, oxidases,decarboxylases, phenoloxidases, ligninases, pullulanases, arabinanases,hemicellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, proteases, peptidases,proteinases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases.

Brewing and Fermenting

The invention provides methods of brewing (e.g., fermenting) beercomprising a glucoamylase and/or an amylase of the invention. In oneexemplary process, starch-containing raw materials are disintegrated andprocessed to form a malt. A glucoamylase and/or an amylase of theinvention can be used at any point in a fermentation process.

For example, amylases and/or glucoamylases of the invention can be usedin the processing of barley malt. The major raw material of beer brewingis barley malt. This can be a three stage process. First, the barleygrain can be steeped to increase water content, e.g., to around about40%. Second, the grain can be germinated by incubation at 15-25° C. for3 to 6 days when enzyme synthesis is stimulated under the control ofgibberellins. During this time amylase levels rise significantly. In oneaspect, amylases and/or glucoamylases of the invention are added at this(or any other) stage of the process. The action of the amylase resultsin an increase in fermentable reducing sugars. This can be expressed asthe diastatic power, DP, which can rise from around 80 to 190 in 5 daysat 12° C.

Amylases and/or glucoamylases of the invention can be used in any beerproducing process, as described, e.g., in U.S. Pat. Nos. 5,762,991;5,536,650; 5,405,624; 5,021,246; 4,788,066.

In one aspect, one or more other enzymes are used in conjunction with afeed, feed or drink comprising enzymes of the invention, e.g., includingother amylases, beta-galactosidases, catalases, laccases, cellulases,endoglycosidases, endo-beta-1,4-laccases, amyloglucosidases, otherglucosidases, glucose isomerases, glycosyltransferases, lipases,phospholipases, lipooxygenases, beta-laccases,endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,glucoamylases, pectinases, reductases, oxidases, decarboxylases,phenoloxidases, ligninases, pullulanases, arabinanases, hemi cellulases,mannanases, xylolaccases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases,polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases,transglutaminases, pectin methylesterases, cellobiohydrolases and/ortransglutaminases.

Use in Drilling Well and Mining Operations

The invention also includes methods using enzymes of the invention inwell and drilling operations, e.g., gas, oil or other drilling or miningoperations. For example, in one aspect, enzymes of the invention areused to increase the flow of production fluids from a subterraneanformation, e.g., a well or a mine. In one aspect, the enzymes of theinvention are used to remove viscous polysaccharide-containing and/orstarch-containing fluids that can be damaging, e.g., fluids formedduring production operations. These polysaccharide-containing and/orstarch-containing fluids can be found within a subterranean formationwhich surrounds a completed well bore. In one aspect, an amylase and/ora glucoamylase of the invention is used in an oil well drilling fluid toaid in the carrying away of drilling mud.

The invention provides methods for changing the viscosity of acomposition comprising: providing a composition and the polypeptide ofthe invention, and a composition; and treating the composition with thepolypeptide of the invention; and in one aspect, of the method, thecomposition comprises a soil or a drilling mud.

In one aspect, the use of these methods of the invention allowsproduction fluids (comprising enzymes of the invention) to flow from thewell bore or a mine. The methods can comprise reducing the flow ofproduction fluids from the formation below expected flow rates andformulating an enzyme treatment by blending together an aqueous fluidand a polypeptide of the invention. The methods can comprise pumping theenzyme treatment to a desired location within the well bore or otherdrilled shaft and allowing the enzyme treatment to degrade the viscous,starch-containing, damaging fluid. The methods can comprise removing thefluid from the subterranean formation to the well or shaft surface. Inone aspect, the enzyme treatment is effective to attack the alphaglucosidic linkages in the starch-containing fluid. In one aspect,amylases and/or glucoamylases of the invention are used in minedrilling, well drilling (e.g., gas or oil well drilling), and the liketo carry away drilling mud, e.g., while drilling the hole (well bore orshaft).

The enzymes of the invention can be used in any well, shaft or minedrilling operation, many of which are well known in the art. Forexample, the invention provides methods of introducing an enzyme of theinvention, which in one aspect can also comprise an oil or gas fieldproduction chemical, into a rock formation comprising oil and/or gas,which comprises passing a microemulsion comprising the enzyme (and, inone aspect, the chemical) down a production well and then into theformation. In one aspect, a production well is subjected to a “shut-in”treatment whereby an aqueous composition comprising an enzyme of theinvention is injected into the production well under pressure and“squeezed” into the formation and held there. See, e.g., U.S. Pat. No.6,581,687.

In one aspect, the amylases and/or glucoamylases of the invention usedin gas, oil or other drilling or mining operations are active at high orlow pH and/or high or low temperatures, e.g., amylases and/orglucoamylases of the invention used in these processes are active underconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4,or lower, or, under conditions comprising about pH 7, pH 7.5 pH 8.0, pH8.5, pH 9, pH 9.5, pH 10, pH 10.5 or pH 11 or higher. In one aspect, theamylases and/or glucoamylases of the invention used in these processesare active under conditions comprising a temperature range of anywherebetween about 0° C. to about 37° C., or, between about 37° C. to about95° C. or more, or, between about 80° C. to about 120° C., e.g., 85° C.,90° C., 95° C., 98° C., 100° C., 105° C., 110° C., 115° C., 120° C. ormore.

In one aspect, one or more other enzymes are used in conjunction with acomposition comprising enzymes of the invention for use with thesemethods, e.g., including other amylases, beta-galactosidases, catalases,laccases, cellulases, endoglycosidases, endo-beta-1,4-laccases,amyloglucosidases, other glucosidases, glucose isomerases,glycosyltransferases, lipases, phospholipases, lipooxygenases,beta-laccases, endo-beta-1,3(4)-laccases, cutinases, peroxidases,amylases, glucoamylases, pectinases, reductases, oxidases,decarboxylases, phenoloxidases, ligninases, pullulanases, arabinanases,hemicellulases, mannanases, xylolaccases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, proteases, peptidases,proteinases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, transglutaminases, pectin methylesterases,cellobiohydrolases and/or transglutaminases.

In one aspect, enzymes or enzyme cocktails of the invention that areused in these gas, oil or other drilling or mining operations, orincluding any oil and gas well washing and/or fracturing processes, areactive at high or low pH and/or high or low temperatures, e.g.,polymer-degrading or polysaccharide-degrading (“polymer breaker”)enzymes of this invention, which include using “cocktails” of these andother enzymes such as amylase, glucoamylase, xanthanase, glycosidaseand/or cellulase enzymes, or a lignin degrading enzyme, alpha amylase,beta amylase, glucoamylase, dextrinase, cellulase, cellobiohydrolase,avicelase, carboxymethylcellulase, beta-glucanase, glucosidase,xylanase, mannanase, arabinofuranosidase, laccase, lignin peroxidase,pectinase, pectate lyase, xanthanase, xanthan lyase, xanthandepolymerase, pullulanase, lichenase, pachymanase, lipase, protease,proteinase, phytase, peptidase and catalase, which include using“cocktails” of these and other enzymes, are used in these processes areactive under conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH4.5, pH 4.0, pH 3.5, pH 3.0 or less (more acidic), or, under conditionscomprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH10.5, pH 11.0, pH 11.5, pH 12, pH 12.5 or more (more basic). In oneaspect, enzymes or enzyme cocktails of the invention used in theseprocesses are active under conditions comprising a temperature range ofanywhere between about −100° C. to about −80° C., about −80° C. to about−40° C., about −40° C. to about −20° C., about −20° C. to about 0° C.,about 0° C. to about 37° C., about 0° C. to about 5° C., about 5° C. toabout 15° C., about 15° C. to about 25° C., about 25° C. to about 37°C., about 37° C. to about 45° C., about 45° C. to about 55° C., about55° C. to about 70° C., about 70° C. to about 75° C., about 75° C. toabout 85° C., about 85° C. to about 90° C., about 90° C. to about 95°C., about 95° C. to about 100° C., about 100° C. to about 105° C., about105° C. to about 110° C., about 110° C. to about 120° C., or 95° C., 96°C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 104° C.,105° C., 106° C., 107° C., 108° C., 109° C., 110° C., 111° C., 112° C.,113° C., 114° C., 115° C., 120° C. or more.

In one embodiment, the “pH trigger mechanism” comprises use of athermophilic enzymes, for example a “pyrolase” such as the polypeptideSEQ ID NO:4 and/or SEQ ID NO:6 (encoded, e.g., by SEQ ID NO:3 and SEQ IDNO:5, respectively). In one aspect, the invention provides a systemcomprising one or more enzymes entrained in a mud or its ingredients ina dry form, e.g., a guar gum powder, sand or a buffer salt. In oneaspect, the enzyme remains dormant or “less active” because of lowtemperature of the mud, or remains dormant or “less active” because theamount of enzyme loading is adjusted such that no or less “premature”(unwanted) degradation (of substrate) occurs until the mud is heated tohigher temperatures conducive to the activation of the enzyme(s). Thisembodiment can be referenced as “an entrained enzyme system with atemperature trigger mechanism”.

In one aspect, the composition and methods of the invention are used todegrade polymers in a “mud”, which in alternative embodiments comprisesa water-containing medium in which enzyme, polysaccharide and othercomponents are mixed. In one aspect, the composition and methods of theinvention are used as a solution or a dry powder, which can be mixedwith an ingredient or component of the “mud”. In one aspect, the enzymeis incorporated/entrained into the ingredients of the mud prior to theactual mud preparation. For example, an exemplary formulation or mixtureused to practice this invention comprises a starch, xanthan or cellulosepowder mixed with one or more enzyme(s), or a mixture of buffer saltsand enzyme(s), wherein each of such enzyme-containing ingredients canthen be used to prepare the mud.

Use of Free and Immobilized Enzymes in Hydraulic Fracturing and DrillingOperations:

The invention provides compositions and methods comprising the inclusionof polymer-breaking (polymer-degrading), e.g., polysaccharide-degrading,enzymes in a free form or in an immobilized form, e.g., in animmobilized form as on a coating, e.g., of a particle, e.g., of a sandgrain or a ceramic material such as a sintered bauxite.

In one aspect, the compositions and methods comprising the inclusion ofpolymer-breaking (polymer-degrading), e.g., polysaccharide-degrading,enzymes in or on a resin or similar material that coats particles, e.g.,sand grains or a ceramic material such as a sintered bauxite; theseparticles (e.g., sand grains) can be used as the proppant or with aproppant (e.g., a resin-coated sand or high-strength ceramic materials)in a hydraulic fracturing fluid. In one aspect, a proppant used topractice this invention is a sized particles mixed with a fracturingfluid to hold fractures open after a hydraulic fracturing treatment. Inaddition to naturally occurring sand grains, man-made or speciallyengineered proppants, such as resin-coated sand or high-strength ceramicmaterials like sintered bauxite, can also be used. Proppant materialscan be sorted for size and sphericity to provide an efficient conduitfor production of fluid from the reservoir to the wellbore. After thesettling of the sand in the well fissures and fractures, the resin-boundenzymes can diffuse out and work on the concentrated and unbrokenpolymer that is often deposited on the formation surface at thecompletion of fracturing operations. Thus, this aspect of the inventioncan effectively remove a polysaccharide, a xanthan or a guar, e.g., aguar filter cake, from fractured oil and gas wells, and/or can enhancethe permeability of the fractured zone.

In one embodiment, during the hydraulic fracturing operations, largevolumes of water, sand, auxiliary chemicals (including enzymes and themixtures of enzymes of this invention) and a polysaccharide-basedpolymer (e.g., a guar and/or its derivatives) are mixed and injectedunder pressure into the oil and/or gas wells to ‘fracture’ thesurrounding formation and enhance the flow of gas or oil into thewellbore. Enzymes and enzyme mixtures as described herein can be used tohydrolyze these polysaccharide polymers and reduce the viscosity of thefluid (used in the hydraulic fracturing operations) for betterpenetration into the formation and more effective flow back at the endof the operation.

In one embodiment, the compositions and methods of this invention areused in enzymatic hydrolysis of base polymers (e.g.,polysaccharide-based polymers, such as guar, xanthan and/or theirderivatives); practicing this invention can solve the problem whereenzymatic hydrolysis of these base polymers may be incomplete to leavesome “unbroken” polymer in the fluid used in the hydraulic fracturingoperations. As the fluid water content is lost to the formation thefluid becomes more concentrated and the unbroken polymers form a thickfilter cake; this filter cake plugs the formation pores and reduces theflow of oil or gas into the wellbore—in one embodiment, the compositionsand methods of this invention are used to break up these filter cakeplugs.

Fracturing fluids contain large amounts of sand, commonly referred to asthe proppant. As the fluid is pumped into the well, the proppant settlesinto the fissures and fractures and prevents them from closing. Thishelps enhance the porosity and permeability of the formation for bettergas/oil flow. The sand grains are often coated with different industrialresins to increase their mechanical strength and prevent them fromcrushing under formation pressure. Thus, in one embodiment, theinvention provides compositions and methods using free or immobilizedpolymer-degrading (“polymer-breaking”) enzymes around, in or on thecoating material of the sand. In one aspect, this is done by entrapmentof the enzyme in the resin or by immobilization on the coating surface.Thus, in this aspect, enzyme(s) used to practice this invention canremain in contact with the filter cake thereby providing continualhydrolysis of the concentrated polymer, removing the cake from thefractures, and enhancing the permeability of the fractured formation.

In on aspect, the invention provides methods using these describedenzyme in drilling operations, e.g., a typical drilling operation, wherea well is created by drilling a hole 5 to 30 inches (13-76 cm) diameterinto the earth with an oil rig, which rotates a drill bit. After thehole is drilled, a steel pipe (casing) slightly smaller than the hole isplaced in the hole, and secured with cement. The casing providesstructural integrity to the newly drilled wellbore in addition toisolating potentially dangerous high pressure zones from each other andfrom the surface.

With these zones safely isolated and the formation protected by thecasing, the well can be drilled deeper (into potentially more-unstableand violent formations) with a smaller bit, and also cased with asmaller size casing. A wells can have 2 to 5 sets of subsequentlysmaller hole sizes drilled inside one another, each cemented withcasing.

To drill the well, the drill bit, aided by rotary torque and thecompressive weight of drill collars above it, breaks up the earth.Drilling fluid, or “mud”, comprising the inclusion of polymer-breaking(polymer-degrading), e.g., polysaccharide-degrading, enzymes and enzymemixtures of this invention, in a free form or in an immobilized form, ispumped down the inside of the drill pipe. The fluid exits at the drillbit and aids to break up the rock, keeping pressure on top of the bit,as well as cleaning, cooling and lubricating the bit.

The generated rock “cuttings” are swept up by the drilling fluid as itcirculates back to surface outside the drill pipe. Fluid comprisingpolymer-breaking (polymer-degrading), e.g., polysaccharide-degrading,enzymes and enzyme mixtures of this invention, in a free form or in animmobilized form, can be added at this stage, too.

The fluids then go over “shakers” which shakes out the cuttings overscreens allowing the good fluid to return back into the pits. Fluidcomprising polymer-breaking (polymer-degrading), e.g.,polysaccharide-degrading, enzymes and enzyme mixtures of this invention,in a free form or in an immobilized form, can be added at this stage,too.

These processes of the invention can be facilitated by addition ofpolymer-breaking (polymer-degrading), e.g., polysaccharide-degrading,enzymes and enzyme mixtures of this invention, in a free form or in animmobilized form. The drilling rig can contain all necessary equipmentto circulate the drilling fluid, hoist and turn the pipe, controldownhole pressures, remove cuttings from the drilling fluid, andgenerate onsite power for these operations.

The enzymes, enzyme mixtures, and methods of the invention can bepracticed with any drilling mud or drilling fluid (some prefer toreserve the term “drilling fluid” for more sophisticated andwell-defined “muds”), or any fluid used in operations to drill boreholesinto the earth. The enzymes, enzyme mixtures, and methods of theinvention can be practiced while drilling oil and/or natural gas wellsand on exploration drilling rigs, including use with simpler holes.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with any well or drillingoperation, e.g., where any mud is used, including use of any of thethree main classification schemes of mud, where “mud” is used broadlyand is separated into 3 categories based on the main component thatmakes up the mud: (1) “Water Based Mud” (WBM), which can be subdividedinto dispersed and non-dispersed muds; (2) “Non Aqueous” or morecommonly “Oil Based Mud” (OBM), including synthetic oils (SBM); and/or(3) Gaseous or Pneumatic mud.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with any well or drillingoperation, e.g., can also be used in or with:

-   -   production wells when they are drilled primarily for producing        oil or gas, once the producing structure and characteristics are        established,    -   appraisal wells when they are used to assess characteristics        (such as flowrate) of a proven hydrocarbon accumulation,    -   exploration wells when they are drilled purely for exploratory        (information gathering) purposes in a new area,    -   wildcat wells when a well is drilled, based on a large element        of hope, in a frontier area where very little is known about the        subsurface.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with any well or drillingoperation, e.g., can also be used in conjunction with methods, equipmentand/or drilling operations as described, e.g., in U.S. PatentApplication Publication No. 20070089910, Hewson, et al., describing,e.g., methods of forming a supported subterranean well bore, and uses,e.g., a positive displacement mud motor.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with methods, equipment and/ordrilling operations as described, e.g., in U.S. Patent ApplicationPublication No. 20070084638, Bohnsack; C., et al., describing, e.g., asystem for facilitating flow of settled solids with drilling fluid froma container, the system including pressure nozzle apparatus with atleast one nozzle from which is flowable fluid under pressure, poweredrotation apparatus for selectively rotating the pressure nozzleapparatus so that the at least one nozzle is movable within thecontainer as fluid is pumped through the at least one nozzle into thecontainer; and, in one aspect, translation apparatus for moving thepressure nozzle apparatus with respect to the container as fluid underpressure is pumped to the at least one rotating nozzle. Mud tanks andmud pits are also described, and the enzymes, enzyme mixtures, andmethods of the invention can be used in or with any of these fluids,and/or in any mud tanks and mud pits used in these types of operations.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with methods, equipment and/ordrilling operations as described, e.g., in U.S. Patent ApplicationPublication No. 20070081157, Csutak; S., et al., describing, e.g.,apparatus for estimating a property of a fluid downhole comprising anultraviolet (UV) light source for inducing light into the fluid at awavelength that produces Raman scattered light at wavelengths that areshorter than wavelengths of substantial fluorescence reflected from thefluid in response to the induced light; a detector that detects aspectrum of the Raman scattered light and provides signals in responseto the detected spectrum; and a processor that processes the signals toprovide an estimate of the a property of the fluid. The enzymes, enzymemixtures, and methods of the invention can be used in or with any ofthese fluids, and/or in operations to estimate filtrate contamination ina formation fluid. For example, these methods include detecting Ramanscatters at a plurality of wavelengths of at least one component presentin an oil-based mud that is not naturally present in the formation, andenzymes, enzyme mixtures, and methods of the invention can be used toaid in the accuracy of this detection.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with methods, equipment and/ordrilling operations as described, e.g., in U.S. Patent ApplicationPublication No. 20070075706, Chen, S., et al., describing, e.g., methodsof evaluating an earth formation comprising making measurements with adownhole tool in a borehole in the earth formation; measuring a Qualityfactor of an antenna of the downhole tool at depths where themeasurements are made; and using the measured Q and a resistivity of amud in the borehole and a formation resistivity, and/or a borehole sizeindicator (BSI), for estimating the other of the formation resistivityand BSI, including measuring the resistivity of the mud in the borehole.The enzymes, enzyme mixtures, and methods of the invention can be usedin or with any of these fluids, and/or in operations to evaluate anearth formation.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with methods, equipment and/ordrilling operations as described, e.g., in U.S. Patent ApplicationPublication No. 20070068675, Barry, M., et al, describing, e.g., methodsfor drilling and completing a gravel packed well, comprising drilling awellbore with a drilling fluid, conditioning the drilling fluid, runningthe gravel packing assembly tools to depth in the wellbore with theconditioned drilling-fluid, and gravel packing a wellbore interval witha completion-fluid. The completion fluid may be the same as thedrilling-fluid. This method may be combined with alternate-path sandscreen technology to ensure proper distribution of the gravel pack. Theproper fluids for drilling, gravel packing and sand screens installationare essential for well completion success. Careful planning, wellpreparation and completion execution are required to increase completionproductivity and longevity. Usually, a minimum of three fluids have beenused to drill and complete gravel packed wells. The first fluid is asolids-laden drilling-fluid used to drill the completion interval. Thesecond fluid is a solids-free completion-fluid used to displace thesolids-laden drilling-fluid and to run sand-exclusion equipment andgravel packing tools in a generally solids-free environment. The thirdfluid is a carrier fluid for the gravel during gravel packing of thecompletion interval. The enzymes, enzyme mixtures, and methods of theinvention can be used in or with any of these fluids (includingsolids-laden drilling-fluids, solids-free completion-fluids and/orcarrier fluids), and/or in operations for drilling and completing agravel packed well.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with methods, equipment and/ordrilling operations as described, e.g., in U.S. Patent ApplicationPublication No. 20070066491, Bicerano; J., et al., use of particles inthe construction, drilling, completion and/or fracture stimulation ofoil and natural gas wells; for example, as a proppant partial monolayer,a proppant pack, an integral component of a gravel pack completion, aball bearing, a solid lubricant, a drilling mud constituent, and/or acement additive, including use of thermoset polymer particles for use inapplications requiring lightweight particles possessing high stiffness,strength, temperature resistance, and/or resistance to aggressiveenvironments. The enzymes, enzyme mixtures, and methods of the inventioncan be used in or with any of these gravel packs, ball bearings, solidlubricants, drilling mud constituents, cement additives and/or thedescribed thermoset polymer particles. The enzymes, enzyme mixtures, andmethods of the invention can be used in or with nanofillers and/ornanocomposites, including heterogeneous nanocomposite morphologies.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with methods, equipment and/ordrilling operations as described, e.g., in U.S. Patent ApplicationPublication No. 20070039735, Robertson; B., et al., describing, e.g.,methods of sealing a permeable zone within a subterranean formation,comprising: preparing a plugging composition comprising oil, clay,magnesium chloride, and magnesium oxide powder; and contacting theplugging composition with water in the subterranean formation such thatthe plugging composition forms a sealing mass, thereby substantiallysealing a permeable zone within the subterranean formation.

The enzymes, enzyme mixtures, and methods of the invention can be usedin, mixed with and/or practiced together with variable density drillingmuds comprising compressible particulate materials, e.g., as describedin U.S. Patent Application Publication No. 20070027036, Polizzotti; R.,et al. The enzymes, enzyme mixtures, and methods of the invention can beused in or with, e.g., drilling muds comprising a compressibleparticulate material in the drilling mud, wherein density of thedrilling mud changes due to a volume change of the compressibleparticulate material in response to pressure or temperature changes andwherein the compressible particulate material is configured to maintainthe density of the drilling mud between a pore pressure gradient and afracture gradient based on the volume change of the compressibleparticulate material in response to pressure changes at certain depths.

The enzymes, enzyme mixtures, and methods of the invention can be usedto modify the viscosity of the drilling mud alone or in conjunction withthe described (see Polizzotti; R., et al.) compressible materials, e.g.,to place the fluid viscosity within pumpability requirements, and/or toadjust the pore pressure gradient and the fracture gradient. Theenzymes, enzyme mixtures, and methods of the invention can be used toeffect a volume change in the drilling mud, e.g., where the drilling mudrheology is configured to achieve a desired composite drilling mudrheology.

In one aspect, the enzymes, enzyme mixtures, and methods of theinvention are used to alter the properties of the drilling mud toprovide a desired composite a mud gel point, e.g., a mud gel point thatcan suspend rock cuttings in an annulus of a wellbore during drillingoperations; and/or to alter the viscosity of the drilling mud inconjunction with, or alone (without), compressible hollow objects (seePolizzotti; R., et al.) to alter pumpability requirements.

In one aspect, the enzymes, enzyme mixtures, and methods of theinvention are used to alter the properties well fluids comprisingdrilling muds, well cleanup fluids, workover fluids, spacer fluids,gravel pack fluids, acidizing fluids and/or fracturing fluids. In oneaspect, the enzymes, enzyme mixtures, and methods of the invention areused to facilitate drilling, completing and/or stimulating asubterranean formation using a variable density fluid, and to modify thevariable density fluid.

In one aspect, the enzymes, enzyme mixtures, and methods of theinvention are used in methods of drilling, completing and/or stimulatingsubterranean formations using a variable density fluid, e.g., bymodifying and/or “adjusting” the density of the fluid; for example, amethod (see Polizzotti; R., et al.) comprising the steps of: introducinga fluid having a density that varies as a function of pressure into thesubterranean formation, where the fluid comprises a base fluid and aportion of elastic particles; and drilling, completing and/orstimulating a subterranean formation using the variable density fluid(which can comprise the enzymes, enzyme mixtures of the invention, orhave been modified by the methods of the invention).

In one aspect, the enzymes, enzyme mixtures, and methods of theinvention are used with the methods and compositions as described inU.S. Pat. No. 4,099,583, describing, e.g., a dual gradient drillingsystem, where a lighter fluid is injected into the mud return annulus(typically in the riser) or other pathway to reduce the mud density fromthe injection point upwards, and the enzymes, enzyme mixtures, andmethods of the invention can modify and/or “adjust” the density of thisfluid.

In one aspect, the enzymes, enzyme mixtures, and methods of theinvention are used with the methods and compositions as described inU.S. Pat. Nos. 6,530,437 and 6,588,501, describing a multi-gradientdrilling method and an apparatus for reduction of hydrostatic pressurein sub sea risers; and U.S. Pat. Nos. 6,422,326, 6,156,708, 5,910,467and 5,881,826, describing the addition of various fluid aphrons todrilling mud formulations.

In one aspect, the enzymes, enzyme mixtures, and methods of theinvention are used with the methods and compositions as described inU.S. Pat. No. 6,497,289, describing use of solid expandable liners,e.g., as tubular systems that are run into a well and expanded.

In alternative embodiments, the enzymes, enzyme mixtures, and methods ofthe invention are used to tailor drilling mud density with depth so thatthe effective mud weight remains between the pore pressure and thefracture gradient at all depths. The required variation in mud densitycan be achieved by changing the properties of fluids with the enzymes,enzyme mixtures, and methods of the invention to modify/change volumeand density, to effect a change in response to pressure. The enzymes,enzyme mixtures, and methods of the invention can be used with anyparticulate components, e.g., various shapes, such as spheres, cubes,pyramids, oblate or prolate spheroids, cylinders, pillows and/or othershapes or structures. The enzymes, enzyme mixtures, and methods of theinvention can be used with any particulate components, e.g.,compressible hollow objects which are filled with pressurized gas, orcompressible solid materials or objects as described in Polizzotti; R.,et al., supra.

In alternative aspects, the enzymes, enzyme mixtures, and methods of theinvention can be used in or with any well or drilling operation, e.g.,including directional drilling, sometimes known as slant drilling, todrill non-vertical wells; including used in any of directional drillingsthree main groups; Oilfield Directional Drilling, Utility InstallationDirectional Drilling (commonly known as H.D.D./Horizontal DirectionalDrilling/Directional boring); and/or in-seam directional drilling(Coal-Bed methane).

In one aspect, the enzymes, enzyme mixtures, and methods of theinvention can be used in conjunction with well logging, a technique usedin the oil and gas industry for recording rock and fluid properties tofind hydrocarbon zones in the geological formations within the Earth'scrust. Logging can be performed to measure the effect of practicing themethods of this invention, e.g., pumping fluids comprising the enzymesor enzyme mixtures of this invention into a well. A logging proceduremay consist of lowering a ‘logging tool’ on the end of a wireline intoan oil well (or hole) to measure the rock and fluid properties of theformation. An interpretation of these measurements is then made tolocate and quantify potential depth zones containing oil and gas(hydrocarbons). Logging tools developed over the years measure theelectrical, acoustic, radioactive, electromagnetic, and other propertiesof the rocks and their contained fluids. Logging is usually performed asthe logging tools are pulled out of the hole. This data is recorded to aprinted record called a ‘Well Log’ and is normally transmitted digitallyto office locations. Well logging is performed at various intervalsduring the drilling of the well and when the total depth is drilled,which could range in depths from 300 m to 8000 m (1000 ft to 25,000 ft)or more.

In addition to the methods, enzymes or enzymes mixtures describedherein, the methods, the enzyme muds or other drilling fluids used topractice this invention can comprise (use of) a water-based drilling mudthat can comprise a bentonite clay (gel), and in some aspects, alsocomprising additives such as barium sulfate (barite), calcium carbonate(chalk) or hematite. Various thickeners also can be used to influencethe viscosity of the fluid, e.g., lignosulfonates, xanthan gum, guargum, glycol, carboxymethylcellulose, polyanionic cellulose (PAC), orstarch. The enzymes or enzymes mixtures described herein, used topractice this invention can be used to modify the properties of (e.g.,the viscosity of) the fluids, e.g., to modify the properties oflignosulfonates, xanthan gum, guar gum, glycol, carboxymethylcellulose,polyanionic cellulose (PAC), or starch.

The methods, enzymes or enzymes mixtures described herein, used topractice this invention can be used to modify the properties ofdeflocculants, which are used to reduce viscosity of clay-based muds;anionic polyelectrolytes, e.g., acrylates, polyphosphates,lignosulfonates (Lig) or tannic acid derivates such as Quebracho (redmud was the name for a Quebracho-based mixture, named after the color ofthe red tannic acid salts; it was commonly used in 1940s to 1950s, thenbecame obsolete when lignosulfates became available).

The methods, enzymes or enzymes mixtures described herein, used topractice this invention can be used in (e.g., added to) water injectorsfor injecting water into a formation, either to maintain reservoirpressure or simply to dispose of water produced with a hydrocarbon(e.g., because even after treatment, it would be too oily and too salineto be considered clean for dumping, e.g., dumping overboard or into afresh water source in the case of onshore wells). Thus, the methods andcompositions (e.g., mixtures of enzymes, immobilized enzymes) of thisinvention are used with water injection as an element of reservoirmanagement and produced water disposal.

The methods, enzymes or enzymes mixtures described herein, used topractice this invention can be used in (e.g., added to) aquiferproducers, e.g., as in intentionally producing reservoir water forre-injection (e.g., in a well bore) to manage pressure; this is ineffect moving reservoir water from where it is not as useful, to whereit is more useful. These wells will generally only be used if producedwater from the oil or gas producer is insufficient for reservoirmanagement purposes. Thus, in one aspect, the methods and compositions(e.g., mixtures of enzymes, immobilized enzymes) of this invention areused with aquifer produced water and/or sea water.

Delayed Release Compositions

The invention provides delayed release or “controlled release”compositions comprising a desired composition coated by a latex polymer,e.g., a latex paint, or equivalent. The delayed release/controlledrelease compositions of the invention can comprise any desiredcomposition, including enzymes or any active ingredient, including smallmolecules, drugs, polysaccharides, lipids, nucleic acids, vitamins,antibiotics, insecticides, and the like. In one aspect, the coating willnot readily dissolve at a relatively low temperature but will decomposeto release the desired composition (e.g., enzyme) at a relatively highertemperature.

The invention provides methods for the delayed release/controlledrelease of compositions wherein the composition is coated by a latexpolymer, e.g., a latex paint, or equivalent.

The delayed release/controlled release compositions and methods of theinvention can be used for a variety of medical and industrialapplications, for example, in one aspect, delayed release/controlledrelease enzyme compositions of the invention comprise enzymes involvedin guar fracturing fluids in enhanced oil recovery operations. Theoilfield guar degrading application of the invention is facilitated by acoating that will not readily dissolve at low temperature but willdecompose to release the enzyme at higher temperatures.

In another aspect, the delayed release/controlled release enzymecompositions of the invention comprise animal feeds or nutritionalsupplements comprising, e.g., enzymes, vitamins, antibiotics and/orother food, drug or nutritional supplements. These active compounds inthe animal feeds or nutritional supplements are protected from pelletingconditions or gastric digestion by the coating on a delayedrelease/controlled release composition of the invention.

In one aspect, the release is a temperature activated release, e.g., thedesired composition (e.g., enzyme) is released at an elevatedtemperature, e.g., between about 37° C. to about 95° C. or more, e.g.,85° C., 90° C., 95° C., 98° C., 100° C. or more. The rate of release canbe controlled by the thickness or amount of “barrier” or latex polymer,applied to the desired composition, e.g., a pellet or matrix comprisingthe desired composition. Thus, the invention provides pellets ormatrices having a range of thicknesses of latex polymer or equivalentand methods of using them.

The invention provides delayed release/controlled release enzymecompositions, e.g., in one aspect, comprising an enzyme of theinvention. In one aspect, the invention provides an enzyme (e.g., anenzyme of the invention), or a pelleted composition comprising an enzyme(e.g., an enzyme of the invention), coated with a latex polymer, e.g., alatex paint, or equivalent. In one aspect, the invention providesmethods of making delayed release enzyme compositions comprising coatingan enzyme (e.g., an enzyme of the invention), or a pelleted compositioncomprising an enzyme (e.g., an enzyme of the invention), with a latexpolymer, e.g., a latex paint, or equivalent. In one aspect, theinvention provides methods of making delayed release/controlled releasecompositions comprising coating a desired compound with a latex polymer,e.g., a latex paint, or equivalent.

Latex polymers that are used in the delayed release/controlled releasecompositions (e.g., delayed release/controlled release enzymecompositions) and methods of the invention include, but are not limitedto, various types such as the following: acrylics; alkyds; celluloses;coumarone-indenes; epoxys; esters; hydrocarbons; maleics; melamines;natural resins; oleo resins; phenolics; polyamides; polyesters; rosins;silicones; styrenes; terpenes; ureas; urethanes; vinyls; and the like.Latex polymers that are used in the delayed release compositions andmethods of the invention also include, but are not limited to, one ormore homo- or copolymers containing one or more of the followingmonomers: (meth)acrylates; vinyl acetate; styrene; ethylene; vinylchloride; butadiene; vinylidene chloride; vinyl versatate; vinylpropionate; t-butyl acrylate; acrylonitrile; neoprene; maleates;fumarates; and the like, including plasticized or other derivativesthereof.

The amount of latex polymer used in the latex composition of theinvention is not critical, but may be any amount following wellestablished procedures using latex polymers. In alternative aspects, theamount of dry latex polymer is at least about 1, or, from about 2 toabout 50, or, from about 3 to about 40 weight percent of the total latexcomposition. The latex composition of the invention may optionallycontain other components such as those generally used in latexcompositions. These additional components include, but are not limitedto, one or more of the following: solvents such as aliphatic or aromatichydrocarbons, alcohols, esters, ketones, glycols, glycol ethers,nitroparaffins or the like; pigments; fillers, dryers; flatting agents;plasticizers; stabilizers; dispersants; surfactants; viscosifiersincluding polymeric associative thickeners, polysaccharide-basedthickeners and so on; suspension agents; flow control agents; defoamers;anti-skinning agents; preservatives; extenders; filming aids;crosslinkers; surface improvers; corrosion inhibitors; and otheringredients useful in latex compositions. In one aspect, latexcompositions of the invention having improved rheology and stability areprovided by combining the latex polymer and a polysaccharide with waterfollowing established procedures. See, e.g., U.S. Pat. Nos. 6,372,901;5,610,225.

In one aspect, in making a pelleted or matrix-comprising composition ofthe invention comprising an active composition, e.g., an enzyme (e.g.,an enzyme of the invention), coated with a latex polymer, e.g., a latexpaint, or equivalent, the active composition (e.g., enzyme) is embeddedin the body of the pellet (in one aspect, a majority, or all, of theactive composition (e.g., enzyme) is embedded in the pellet. Thus, harshchemicals, e.g., the latex coating, which may be an inactivator of thedesired, active ingredient, can be used to coat the surface of thepellet or matrix. The composition of the coating can be broken down byagents such as heat, acid, base, pressure, enzymes, other chemicals andthe like, to have a controlled release of the desired enzymatic activitytriggered by the exposure to the coating-degrading agent.

In one aspect, an active composition, e.g., an enzyme (e.g., an enzymeof the invention, or another enzyme, e.g., a mannanase), is dispersed ina corn term meal and/or a corn starch matrix (e.g., as a pellet). Thismixture (e.g., pellet) disintegrates within ten minutes in roomtemperature (e.g., about 22° C.) water to release all (100%) of theactive composition, e.g., releases all of the enzymatic activity. Athigher temperatures, the rate of release increases. This is not anacceptable rate of disintegration for many uses.

However, as a delayed release/controlled release composition of theinvention, i.e., when this mixture is coated with a latex polymer, e.g.,a latex paint, or equivalent, the disintegration of the mixture (e.g.,pellet, matrix) is delayed. The rate and extent of release can becontrolled by the thickness of the coating (barrier) applied to thepellet or matrix. For example, a coated particle will release only 30%of the activity after six hours in 22° C. water. At 60° C., 50% of theenzyme is released in 90 minutes. At 80° C., 80% of the enzyme isreleased during one hour.

In one aspect, one or more other enzymes are added to a delayedrelease/controlled release composition of the invention, e.g., includingother amylases, beta-galactosidases, catalases, laccases, cellulases,endoglycosidases, endo-beta-1,4-laccases, amyloglucosidases, otherglucosidases, glucose isomerases, glycosyltransferases, lipases,phospholipases, lipooxygenases, beta-laccases,endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,glucoamylases, pectinases, reductases, oxidases, decarboxylases,phenoloxidases, ligninases, pullulanases, arabinanases, hemi cellulases,mannanases, xylolaccases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases,polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases,transglutaminases, pectin methylesterases, cellobiohydrolases and/ortransglutaminases.

Biomass Conversion and Production of Clean Bio Fuels

The invention provides enzymes and methods for the conversion of biomassto fuels (e.g., bioethanol, biopropanol, biobutanol, or biodiesel) andchemicals. Thus, the compositions and methods of the invention provideeffective and sustainable alternatives to use of petroleum-basedproducts. The invention provides organisms expressing enzymes of theinvention for participation in chemical cycles involving natural biomassconversion. In one aspect, enzymes and methods for the conversion areused in enzyme ensembles for the efficient depolymerization of biomasspolymers to metabolizable carbon moieties. As discussed above, theinvention provides methods for discovering and implementing the mosteffective of enzymes to enable these important new “biomass conversion”and alternative energy industrial processes.

In one aspect, the polypeptides of the invention are used in processesfor converting lignocellulosic and/or starch biomass to ethanol. Theinvention also provides processes for making ethanol (“bioethanol”),propanol (“biopropanol”), butanol (“biobutanol”), or diesel fuel(“biodiesel”), from compositions comprising starch and/orlignocellulosic biomass. The lignocellulose and/or starch biomassmaterial can be obtained from agricultural crops, as a byproduct of foodor feed production, or as lignocellulosic waste products, such as plantresidues and waste paper. Examples of suitable plant residues fortreatment with polypeptides of the invention include stems, leaves,hulls, husks, cobs and the like, as well as wood, wood chips, wood pulp,and sawdust. Examples of paper waste suitable for treatment withpolypeptides of the invention include discard photocopy paper, computerprinter paper, notebook paper, notepad paper, typewriter paper, and thelike, as well as newspapers, magazines, cardboard, and paper-basedpackaging materials.

In one aspect, the enzymes and methods of the invention can be used inconjunction with more “traditional” means of making ethanol frombiomass, e.g., as methods comprising hydrolyzing lignocellulosicmaterials by subjecting dried lignocellulosic material in a reactor to acatalyst comprised of a dilute solution of a strong acid and a metalsalt; this can lower the activation energy, or the temperature, ofcellulose hydrolysis to obtain higher sugar yields; see, e.g., U.S. Pat.Nos. 6,660,506; 6,423,145.

Another exemplary method that incorporates use of enzymes of theinvention comprises catalyzing the hydrolysis of polysaccharidescomprising glucose monomers, such as starch (a polymer of glucosemonomers joined by 1,4-alpha or 1,6-alpha linkages), into sugars; thiscan be used in conjunction with enzyme for hydrolyzing lignocellulosicmaterial containing hemicellulose, cellulose and lignin. In one aspect,the biomass is subjected to a first stage hydrolysis step in an aqueousmedium at a temperature and a pressure chosen to effect primarilydepolymerization of hemicellulose without major depolymerization ofcellulose to glucose. This step results in a slurry in which the liquidaqueous phase contains dissolved monosaccharides resulting fromdepolymerization of hemicellulose and a solid phase containing celluloseand lignin. A second stage hydrolysis step can comprise conditions suchthat at least a major portion of the cellulose is depolymerized, suchstep resulting in a liquid aqueous phase containing dissolved/solubledepolymerization products of cellulose, which can be hydrolyzed withenzymes of this invention. See, e.g., U.S. Pat. No. 5,536,325. Enzymesof the invention can be added at any stage of this exemplary process.

Another exemplary method incorporating enzymes of the inventioncomprises processing a lignocellulose-containing biomass material by oneor more stages of dilute acid hydrolysis with about 0.4% to 2% strongacid; and treating an unreacted solid lignocellulosic component of theacid hydrolyzed biomass material by alkaline delignification to produceprecursors for biodegradable thermoplastics and derivatives. See, e.g.,U.S. Pat. No. 6,409,841. Enzymes of the invention can be added at anystage of this exemplary process.

Another exemplary method that incorporated use of enzymes of theinvention comprises prehydrolyzing lignocellulosic material in aprehydrolysis reactor; adding an acidic liquid to the solidlignocellulosic material to make a mixture; heating the mixture toreaction temperature; maintaining reaction temperature for timesufficient to fractionate the lignocellulosic material into asolubilized portion containing at least about 20% of the lignin from thelignocellulosic material and a solid fraction containing cellulose;removing a solubilized portion from the solid fraction while at or nearreaction temperature wherein the cellulose in the solid fraction isrendered more amenable to enzymatic digestion; and recovering asolubilized portion. See, e.g., U.S. Pat. No. 5,705,369. Enzymes of theinvention can be added at any stage of this exemplary process.

The invention provides methods for making motor fuel compositions (e.g.,for spark ignition motors) based on liquid hydrocarbons blended with afuel grade alcohol made by using an enzyme or a method of the invention.In one aspect, the fuels made by use of an enzyme of the inventioncomprise, e.g., coal gas liquid- or natural gas liquid-ethanol blends.In one aspect, a co-solvent is biomass-derived 2-methyltetrahydrofuran(MTHF). See, e.g., U.S. Pat. No. 6,712,866.

Methods of the invention for the enzymatic degradation oflignocellulose, e.g., for production of ethanol from lignocellulosicmaterial, can also comprise use of ultrasonic treatment of the biomassmaterial; see, e.g., U.S. Pat. No. 6,333,181.

Another exemplary process for making a biofuel comprising, e.g., abioethanol, biopropanol, biobutanol, or a biodiesel, using enzymes ofthe invention comprises pretreating a starting material comprising alignocellulosic feedstock comprising at least hemicellulose andcellulose. In one aspect, the starting material comprises potatoes,soybean (rapeseed), barley, rye, corn, oats, wheat, beets or sugar caneor a component or waste or food or feed production byproduct. Thestarting material (“feedstock”) is reacted at conditions which disruptthe plant's fiber structure to effect at least a partial hydrolysis ofthe hemicellulose and cellulose. Disruptive conditions can comprise,e.g., subjecting the starting material to an average temperature of 180°C. to 270° C. at pH 0.5 to 2.5 for a period of about 5 seconds to 60minutes; or, temperature of 220° C. to 270° C., at pH 0.5 to 2.5 for aperiod of 5 seconds to 120 seconds, or equivalent. This generates afeedstock with increased accessibility to being digested by an enzyme,e.g., an amylase or a glucoamylase, of this invention. U.S. Pat. No.6,090,595.

Exemplary conditions for enzyme hydrolysis of lignocellulosic materialinclude reactions at temperatures between about 30° C. and 48° C.,and/or a pH between about 4.0 and 6.0. Other exemplary conditionsinclude a temperature between about 30° C. and 60° C. and a pH betweenabout 4.0 and 8.0.

In one aspect of these biofuel (such as a bioethanol, biopropanol,biobutanol, or a biodiesel) generating processes of the invention usingat least one enzyme of the invention, one or more other enzymes areadded, e.g., other amylases, beta-galactosidases, catalases, laccases,cellulases, endoglycosidases, endo-beta-1,4-laccases, amyloglucosidases,other glucosidases, glucose isomerases, glycosyltransferases, lipases,phospholipases, lipooxygenases, beta-laccases,endo-beta-1,3(4)-laccases, cutinases, peroxidases, amylases,glucoamylases, pectinases, reductases, oxidases, decarboxylases,phenoloxidases, ligninases, pullulanases, arabinanases, hemi cellulases,mannanases, xylolaccases, xylanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, proteases, peptidases, proteinases,polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases,transglutaminases, pectin methylesterases, cellobiohydrolases and/ortransglutaminases.

Thus, the invention provides methods for processing a biomass materialcomprising lignocellulose comprising contacting a composition comprisinga polypeptide of the invention, wherein optionally the biomass materialcomprises or is derived from an agricultural crop, or is a byproduct ofa food or a feed production, or is a lignocellulosic waste product, oris a plant residue or a waste paper or waste paper product, andoptionally the polypeptide has activity comprising amylase,glucoamylase, glucosidase, e.g. alpha-glucosidase or beta-glucosidaseactivity, and optionally the plant residue comprise stems, leaves,hulls, husks, cobs, wood, wood chips, wood pulp and sawdust, andoptionally the paper waste comprises discarded or used photocopy paper,computer printer paper, notebook paper, notepad paper, typewriter paper,newspapers, magazines, cardboard and paper-based packaging materials,and optionally the processing of the biomass material generates abioethanol. The invention provides biomass material comprising apolypeptide of the invention.

The invention provides methods for making biofuel (such as a bioethanol,biopropanol, biobutanol, or a biodiesel) comprising contacting acomposition comprising a fermentable sugar with a polypeptide of theinvention, wherein optionally the composition comprising a fermentablesugar comprises a plant, plant product or plant derivative, andoptionally the plant or plant product comprises cane sugar plants orplant products, beets or sugarbeets, wheat, corn, soybeans, potato, riceor barley, and optionally the polypeptide has activity comprisingamylase, glucoamylase, glucosidase, e.g. alpha-glucosidase orbeta-glucosidase activity.

The invention provides methods for making a fuel (such as a bioethanol,biopropanol, biobutanol, or a biodiesel) comprising contacting acomposition comprising a fermentable sugar with a polypeptide of theinvention, wherein optionally the composition comprising a fermentablesugar comprises a plant, plant product or plant derivative, andoptionally the plant or plant product comprises cane sugar plants orplant products, beets or sugarbeets, wheat, corn, soybeans, potato, riceor barley, and optionally the polypeptide has activity comprisingamylase, glucoamylase, glucosidase, e.g. alpha-glucosidase orbeta-glucosidase activity, and optionally the fuel comprises abioethanol or a gasoline-ethanol mix. The invention provides fuelscomprising a polypeptide of the invention, wherein optionally thepolypeptide has activity comprising amylase, glucoamylase, glucosidase,e.g. alpha-glucosidase or beta-glucosidase activity, wherein optionallythe fuel is derived from a plant material, which optionally comprisespotatoes, soybean (rapeseed), barley, rye, corn, oats, wheat, beets orsugar cane, and optionally the fuel comprises a bioethanol, biopropanol,biobutanol, biodiesel and/or a gasoline-ethanol mix.

In another aspect, plant material comprising the enzymes describedherein can be used in an industrial process to produce fuel or energy.Enzymes expressed in plants can be added to, mixed into or sprayed ontofeedstock material. Alternatively, the enzymes could be directlyexpressed in the feedstock material. In one embodiment, plant materialexpressing enzymes could be ground, milled, heated or the like, in orderto disrupt the physical integrity of the plant cells or organs thatcontain the enzyme, thereby releasing the enzyme to come in contact withthe substrate. Exemplary sources of plant material include, but are notlimited to, maize, alfalfa, sunflower, Brassica, soybean, cotton,safflower, peanut, sorghum, wheat, oat, rye, millet, barley, rice,conifers, grasses, e.g., switch grass and Miscanthus, legume crops,e.g., pea, bean and soybean, starchy tuber/roots, e.g., potato, sweetpotato, cassava, taro, canna and sugar beet and the like. The inventionprovides polypeptide, including amylases and/or glucoamylases of theinvention and antibodies, and methods for the conversion of a biomass orany lignocellulosic material (e.g., any composition comprisingcellulose, hemicellulose and lignin), to a fuel (e.g., bioethanol,biopropanol, biobutanol, biopropanol, biomethanol, biodiesel), inaddition to feeds, foods and chemicals. For example, in one aspect, anenzyme of the invention has β-glucosidase activity to liberate D-glucosefrom cellobiose dimers. In one aspect, the enzymes have exo- orendo-beta-glucanase activity.

Thus, the compositions and methods of the invention provide effectiveand sustainable alternatives or adjuncts to use of petroleum-basedproducts, e.g., as a mixture of a biofuel such as biomethanol,bioethanol, biopropanol, biobutanol, and the like, to diesel fuel,gasoline, kerosene and the like. The invention provides organismsexpressing enzymes of the invention for participation in chemical cyclesinvolving natural biomass conversion. In one aspect, enzymes and methodsfor the conversion are used in enzyme ensembles for the efficientdepolymerization of polysaccharides, cellulosic and/or hemicellulosicpolymers to metabolizeable (e.g., fermentable) carbon moieties. Theinvention provides methods for discovering and implementing the mosteffective of enzymes to enable these important new “biomass conversion”and alternative energy industrial processes.

The compositions and methods of the invention can be used to provideeffective and sustainable alternatives or adjuncts to use ofpetroleum-based products, e.g., as a mixture of bioethanol, biopropanol,biobutanol, biopropanol, biomethanol and/or biodiesel and gasoline. Theinvention provides organisms expressing enzymes of the invention forparticipation in chemical cycles involving natural biomass conversion.The invention provides methods for discovering and implementing the mosteffective of enzymes to enable these important new “biomass conversion”and alternative energy industrial processes.

The invention provides methods, enzymes and mixtures of enzymes or“cocktails” of the invention, for processing a material, e.g. a biomassmaterial, comprising a cellooligsaccharide, an arabinoxylan oligomer, alignin, a lignocellulose, a xylan, a glucan, a cellulose and/or afermentable sugar comprising contacting the composition with apolypeptide of the invention, or a polypeptide encoded by a nucleic acidof the invention, wherein optionally the material is derived from anagricultural crop (e.g., wheat, barley, potatoes, switchgrass, poplarwood), is a byproduct of a food or a feed production, is alignocellulosic waste product, or is a plant residue or a waste paper orwaste paper product, and optionally the plant residue comprise stems,leaves, hulls, husks, corn or corn cobs, corn stover, corn fiber, hay,straw (e.g. rice straw or wheat straw), sugarcane bagasse, sugar beetpulp, citrus pulp, and citrus peels, wood, wood thinnings, wood chips,wood pulp, pulp waste, wood waste, wood shavings and sawdust,construction and/or demolition wastes and debris (e.g. wood, woodshavings and sawdust), and optionally the paper waste comprisesdiscarded or used photocopy paper, computer printer paper, notebookpaper, notepad paper, typewriter paper, newspapers, magazines, cardboardand paper-based packaging materials, and recycled paper materials. Inaddition, urban wastes, e.g. the paper fraction of municipal solidwaste, municipal wood waste, and municipal green waste, along with othermaterials containing sugar, starch, and/or cellulose can be used.Optionally the processing of the material, e.g. the biomass material,generates a bioalcohol, e.g., a bioethanol, biomethanol, biobutanol orbiopropanol.

Alternatively, the polypeptide of the invention may be expressed in thebiomass plant material or feedstock itself.

The methods of the invention also include taking the convertedlignocellulosic material (processed by enzymes of the invention) andmaking it into a fuel (e.g. a bioalcohol, e.g., a bioethanol,biomethanol, biobutanol or biopropanol, or biodiesel) by fermentationand/or by chemical synthesis. In one aspect, the produced sugars arefermented and/or the non-fermentable products are gasified.

The methods of the invention also include converting algae, virginvegetable oils, waste vegetable oils, animal fats and greases (e.g.tallow, lard, and yellow grease), or sewage, using enzymes of theinvention, and making it into a fuel (e.g. a bioalcohol, e.g., abioethanol, biomethanol, biobutanol or biopropanol, or biodiesel) byfermentation and/or by chemical synthesis or conversion.

The enzymes of the invention (including, for example, organisms, such asmicroorganisms, e.g., fungi, yeast or bacteria, making and in someaspects secreting recombinant enzymes of the invention) can be used inor included/integrated at any stage of any biomass conversion process,e.g., at any one step, several steps, or included in all of the steps,or all of the following methods of biomass conversion processes, or allof these biofuel alternatives:

Direct combustion: the burning of material by direct heat and is thesimplest biomass technology; can be very economical if a biomass sourceis nearby.

Pyrolysis: is the thermal degradation of biomass by heat in the absenceof oxygen. In one aspect, biomass is heated to a temperature betweenabout 800 and 1400 degrees Fahrenheit, but no oxygen is introduced tosupport combustion resulting in the creation of gas, fuel oil andcharcoal.

Gasification: biomass can be used to produce methane through heating oranaerobic digestion. Syngas, a mixture of carbon monoxide and hydrogen,can be derived from biomass.

Landfill Gas: is generated by the decay (anaerobic digestion) of buriedgarbage in landfills. When the organic waste decomposes, it generatesgas consisting of approximately 50% methane, the major component ofnatural gas.

Anaerobic digestion: converts organic matter to a mixture of methane,the major component of natural gas, and carbon dioxide. In one aspect,biomass such as waterwaste (sewage), manure, or food processing waste,is mixed with water and fed into a digester tank without air.

Fermentation

Alcohol Fermentation: fuel alcohol is produced by converting cellulosicmass and/or starch to sugar, fermenting the sugar to alcohol, thenseparating the alcohol water mixture by distillation. Feedstocks such asdedicated crops (e.g., wheat, barley, potatoes, switchgrass, poplarwood), agricultural residues and wastes (e.g. rice straw, corn stover,wheat straw, sugarcane bagasse, rice hulls, corn fiber, sugar beet pulp,citrus pulp, and citrus peels), forestry wastes (e.g. hardwood andsoftwood thinnings, hardwood and softwood residues from timberoperations, wood shavings, and sawdust), urban wastes (e.g. paperfraction of municipal solid waste, municipal wood waste, municipal greenwaste), wood wastes (e.g. saw mill waste, pulp mill waste, constructionwaste, demolition waste, wood shavings, and sawdust), and waste paper orother materials containing sugar, starch, and/or cellulose can beconverted to sugars and then to alcohol by fermentation with yeast.Alternatively, materials containing sugars can be converted directly toalcohol by fermentation.

Transesterification: An exemplary reaction for converting oil tobiodiesel is called transesterification. The transesterification processreacts an alcohol (like methanol) with the triglyceride oils containedin vegetable oils, animal fats, or recycled greases, forming fatty acidalkyl esters (biodiesel) and glycerin. The reaction requires heat and astrong base catalyst, such as sodium hydroxide or potassium hydroxide.

Biodiesel: Biodiesel is a mixture of fatty acid alkyl esters made fromvegetable oils, animal fats or recycled greases. Biodiesel can be usedas a fuel for vehicles in its pure form, but it is usually used as apetroleum diesel additive to reduce levels of particulates, carbonmonoxide, hydrocarbons and air toxics from diesel-powered vehicles.

Hydrolysis: includes hydrolysis of a compound, e.g., a biomass, such asa lignocellulosic material, catalyzed using an enzyme of the instantinvention.

Congeneration: is the simultaneous production of more than one form ofenergy using a single fuel and facility. In one aspect, biomasscogeneration has more potential growth than biomass generation alonebecause cogeneration produces both heat and electricity.

In one aspect, the polypeptides of the invention have enzymatic activity(including, e.g., an amylase or a glucoamylase activity) for generatinga fuel (e.g. a bioalcohol, e.g., a bioethanol, biomethanol, biobutanolor biopropanol, or biodiesel) from an organic material, e.g., a biomass,such as compositions derived from plants and animals, including anyagricultural crop or other renewable feedstock, an agricultural residueor an animal waste, the organic components of municipal and industrialwastes, or construction or demolition wastes or debris, ormicroorganisms such as algae or yeast.

In one aspect, polypeptides of the invention are used in processes forconverting lignocellulosic biomass to a fuel (e.g. a bioalcohol, e.g., abioethanol, biomethanol, biobutanol or biopropanol, or biodiesel), orotherwise are used in processes for hydrolyzing or digestingbiomaterials such that they can be used as a fuel (e.g. a bioalcohol,e.g., a bioethanol, biomethanol, biobutanol or biopropanol, orbiodiesel), or for making it easier for the biomass to be processed intoa fuel.

In an alternative aspect, polypeptides of the invention, including themixture of enzymes or “cocktails” of the invention, are used inprocesses for a transesterification process reacting an alcohol (likeethanol, propanol, butanol, propanol, methanol) with a triglyceride oilcontained in a vegetable oil, animal fat or recycled greases, formingfatty acid alkyl esters (biodiesel) and glycerin. In one aspect,biodiesel is made from soybean oil or recycled cooking oils. Animal'sfats, other vegetable oils, and other recycled oils can also be used toproduce biodiesel, depending on their costs and availability. In anotheraspect, blends of all kinds of fats and oils are used to produce abiodiesel fuel of the invention.

Enzymes of the invention, including the mixture of enzymes or“cocktails” of the invention, can also be used in glycerin refining. Theglycerin by-product contains unreacted catalyst and soaps that areneutralized with an acid. Water and alcohol are removed to produce 50%to 80% crude glycerin. The remaining contaminants include unreacted fatsand oils, which can be processes using the polypeptides of theinvention. In a large biodiesel plants of the invention, the glycerincan be further purified, e.g., to 99% or higher purity, for thepharmaceutical and cosmetic industries.

Fuels (including bioalcohols such as bioethanols, biomethanols,biobutanols or biopropanols, or biodiesels) made using the polypeptidesof the invention, including the mixture of enzymes or “cocktails” of theinvention, can be used with fuel oxygenates to improve combustioncharacteristics. Adding oxygen results in more complete combustion,which reduces carbon monoxide emissions. This is another environmentalbenefit of replacing petroleum fuels with biofuels (e.g., a fuel of theinvention). A biofuel made using the compositions and/or methods of thisinvention can be blended with gasoline to form an Eli) blend (about 5%to 10% ethanol and about 90% to 95% gasoline), but it can be used inhigher concentrations such as E85 or in its pure form. A biofuel madeusing the compositions and/or methods of this invention can be blendedwith petroleum diesel to form a B20 blend (20% biodiesel and 80%petroleum diesel), although other blend levels can be used up to B100(pure biodiesel).

The invention also provides processes for making biofuels (includingbioalcohols such as bioethanols, biomethanols, biobutanols orbiopropanols, or biodiesels) from compositions comprisinglignocellulosic biomass. The lignocellulose biomass material can beobtained from agricultural crops, as a byproduct of food or feedproduction, or as lignocellulosic waste products, such as plantresidues, waste paper or construction and/or demolition wastes ordebris. Examples of suitable plant sources or plant residues fortreatment with polypeptides of the invention include kelp, algae,grains, seeds, stems, leaves, hulls, husks, corn cobs, corn stover,straw, grasses (e.g., Indian grass, such as Sorghastrum nutans; or,switch grass, e.g., Panicum species, such as Panicum virgatum), and thelike, as well as wood, wood chips, wood pulp, and sawdust. Examples ofpaper waste suitable for treatment with polypeptides of the inventioninclude discard photocopy paper, computer printer paper, notebook paper,notepad paper, typewriter paper, and the like, as well as newspapers,magazines, cardboard, and paper-based packaging materials. Examples ofconstruction and demolition wastes and debris include wood, wood scraps,wood shavings and sawdust.

In one embodiment, the enzymes, including the mixture of enzymes or“cocktails” of the invention, and methods of the invention can be usedin conjunction with more “traditional” means of making ethanol,methanol, propanol, butanol, propanol and/or diesel from biomass, e.g.,as methods comprising hydrolyzing lignocellulosic materials bysubjecting dried lignocellulosic material in a reactor to a catalystcomprised of a dilute solution of a strong acid and a metal salt; thiscan lower the activation energy, or the temperature, of cellulosehydrolysis to obtain higher sugar yields; see, e.g., U.S. Pat. Nos.6,660,506 and 6,423,145.

Another exemplary method that incorporated use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises hydrolyzing lignocellulosic material containinghemicellulose, cellulose and lignin, or any other polysaccharide thatcan be hydrolyzed by an enzyme of this invention, by subjecting thematerial to a first stage hydrolysis step in an aqueous medium at atemperature and a pressure chosen to effect primarily depolymerizationof hemicellulose without major depolymerization of cellulose to glucose.This step results in a slurry in which the liquid aqueous phase containsdissolved monosaccharides resulting from depolymerization ofhemicellulose and a solid phase containing cellulose and lignin. Asecond stage hydrolysis step can comprise conditions such that at leasta major portion of the cellulose is depolymerized, such step resultingin a liquid aqueous phase containing dissolved/soluble depolymerizationproducts of cellulose. See, e.g., U.S. Pat. No. 5,536,325. Enzymes ofthe invention (including the invention's mixtures, or “cocktails” ofenzymes) can be added at any stage of this exemplary process.

Another exemplary method that incorporated use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises processing a lignocellulose-containing biomassmaterial by one or more stages of dilute acid hydrolysis with about 0.4%to 2% strong acid; and treating an unreacted solid lignocellulosiccomponent of the acid hydrolyzed biomass material by alkalinedelignification to produce precursors for biodegradable thermoplasticsand derivatives. See, e.g., U.S. Pat. No. 6,409,841. Enzymes of theinvention can be added at any stage of this exemplary process.

Another exemplary method that incorporated use of enzymes of theinvention, including the mixture of enzymes or “cocktails” of theinvention, comprises prehydrolyzing lignocellulosic material in aprehydrolysis reactor; adding an acidic liquid to the solidlignocellulosic material to make a mixture; heating the mixture toreaction temperature; maintaining reaction temperature for timesufficient to fractionate the lignocellulosic material into asolubilized portion containing at least about 20% of the lignin from thelignocellulosic material and a solid fraction containing cellulose;removing a solubilized portion from the solid fraction while at or nearreaction temperature wherein the cellulose in the solid fraction isrendered more amenable to enzymatic digestion; and recovering asolubilized portion. See, e.g., U.S. Pat. No. 5,705,369. Enzymes of theinvention can be added at any stage of this exemplary process.

The invention provides methods for making motor fuel compositions (e.g.,for spark ignition motors) based on liquid hydrocarbons blended with afuel grade alcohol made by using an enzyme or a method of the invention.In one aspect, the fuels made by use of an enzyme of the inventioncomprise, e.g., coal gas liquid- or natural gas liquid-ethanol blends.In one aspect, a co-solvent is biomass-derived 2-methyltetrahydrofuran(MTHF). See, e.g., U.S. Pat. No. 6,712,866.

In one aspect, methods of the invention for the enzymatic degradation oflignocellulose, e.g., for production of biofuels (including bioalcoholssuch as bioethanols, biomethanols, biobutanols or biopropanols, orbiodiesels) from lignocellulosic material, can also comprise use ofultrasonic treatment of the biomass material; see, e.g., U.S. Pat. No.6,333,181.

In another aspect, methods of the invention for producing biofuels(including bioalcohols such as bioethanols, biomethanols, biobutanols orbiopropanols, or biodiesels) from a cellulosic substrate compriseproviding a reaction mixture in the form of a slurry comprisingcellulosic substrate, an enzyme of this invention and a fermentationagent (e.g., within a reaction vessel, such as a semi-continuouslysolids-fed bioreactor), and the reaction mixture is reacted underconditions sufficient to initiate and maintain a fermentation reaction(as described, e.g., in U.S. Pat. App. No. 20060014260). In one aspect,experiment or theoretical calculations can determine an optimum feedingfrequency. In one aspect, additional quantities of the cellulosicsubstrate and the enzyme are provided into the reaction vessel at aninterval(s) according to the optimized feeding frequency.

One exemplary process for making biofuels (including bioalcohols such asbioethanols, biomethanols, biobutanols or biopropanols, or biodiesels)of the invention is described in U.S. Pat. App. Pub. Nos. 20050069998;20020164730; and in one aspect comprises stages of grinding thelignocellulosic biomass (e.g., to a size of 15-30 mm), subjecting theproduct obtained to steam explosion pre-treatment (e.g., at atemperature of 190-230° C.) for between 1 and 10 minutes in a reactor;collecting the pre-treated material in a cyclone or related product ofmanufacture; and separating the liquid and solid fractions by filtrationin a filter press, introducing the solid fraction in a fermentationdeposit and adding one or more enzymes of the invention, e.g., anamylase, glucoamylase, and/or glucosidase enzyme (e.g., dissolved incitrate buffer pH 4.8).

Another exemplary process for making biofuels (including bioalcoholssuch as bioethanols, biomethanols, biobutanols or biopropanols, orbiodiesels) of the invention comprising bioethanols, biomethanols,biobutanols or biopropanols using enzymes of the invention comprisespretreating a starting material comprising a lignocellulosic feedstockcomprising at least hemicellulose and cellulose. In one aspect, thestarting material comprises potatoes, soybean (rapeseed), barley, rye,corn, oats, wheat, beets or sugar cane or a component or waste or foodor feed production byproduct. The starting material (“feedstock”) isreacted at conditions which disrupt the plant's fiber structure toeffect at least a partial hydrolysis of the hemicellulose and cellulose.Disruptive conditions can comprise, e.g., subjecting the startingmaterial to an average temperature of 180° C. to 270° C. at pH 0.5 to2.5 for a period of about 5 seconds to 60 minutes; or, temperature of220° C. to 270° C., at pH 0.5 to 2.5 for a period of 5 seconds to 120seconds, or equivalent. This generates a feedstock with increasedaccessibility to being digested by an enzyme, e.g., a cellulase enzymeof the invention. U.S. Pat. No. 6,090,595.

Exemplary conditions for using enzymes of the invention in thehydrolysis of lignocellulosic material include reactions at temperaturesbetween about 30° C. and 48° C., and/or a pH between about 4.0 and 6.0.Other exemplary conditions include a temperature between about 30° C.and 60° C. and a pH between about 4.0 and 8.0.

Amylases and/or glucoamylases of the invention can be used in theconversion of biomass to fuels, and in the production of ethanol, e.g.,as described in PCT Application Nos. WO0043496 and WO8100857. Amylasesand/or glucoamylases of the invention can be used to produce fermentablesugars and glucan-containing biomass that can be converted into fuelethanol.

Pharmaceutical Compositions, Disinfectants and Dietary Supplements

The invention also provides pharmaceutical compositions, disinfectantsand dietary supplements (e.g., dietary aids) comprising enzymes of theinvention (e.g., enzymes having amylase, glucoamylase or glucosidaseactivity). In one aspect, the pharmaceutical compositions and dietarysupplements (e.g., dietary aids) are formulated for oral ingestion,e.g., to improve the digestibility of foods and feeds having a highstarch, cellulose or lignocellulosic component.

Periodontal treatment compounds can comprise an enzyme of the invention,e.g., as described in U.S. Pat. No. 6,776,979. Compositions and methodsfor the treatment or prophylaxis of acidic gut syndrome can comprise anenzyme of the invention, e.g., as described in U.S. Pat. No. 6,468,964.

In another aspect, wound dressings, implants and the like compriseantimicrobial (e.g., antibiotic-acting) enzymes, including an enzyme ofthe invention. Enzymes of the invention can also be used in alginatedressings, antimicrobial barrier dressings, burn dressings, compressionbandages, diagnostic tools, gel dressings, hydro-selective dressings,hydrocellular (foam) dressings, hydrocolloid dressings, I.V dressings,incise drapes, low adherent dressings, odor absorbing dressings, pastebandages, post operative dressings, scar management, skin care,transparent film dressings and/or wound closure. Enzymes of theinvention can be used in wound cleansing, wound bed preparation, totreat pressure ulcers, leg ulcers, burns, diabetic foot ulcers, scars,IV fixation, surgical wounds and minor wounds. Enzymes of the inventioncan be used to in sterile enzymatic debriding compositions, e.g.,ointments. In various aspects, the cellulase is formulated as a tablet,gel, pill, implant, liquid, spray, powder, food, feed pellet or as anencapsulated formulation.

Biodefense Applications

In other aspects, enzymes of the invention, e.g., amylases,glucoamylases or glucosidases, can be used in biodefense, e.g.,destruction of spores or bacteria. Use of enzymes of the invention inbiodefense applications offer a significant benefit, in that they can bevery rapidly developed against any currently unknown or biologicalwarfare agents of the future. In addition, enzymes of the invention canbe used for decontamination of affected environments. In aspect, theinvention provides a biodefense or bio-detoxifying agent comprising apolypeptide of the invention having amylase, glucoamylase or glucosidaseactivity.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

Examples Example 1: Identification and Characterization of Thermostableα-Amylases

The following example describes exemplary methods for determining if apolypeptide is within the scope of the invention. Screening programs canbe carried out under neutral and low pH conditions. DNA sequence andbioinformatic analyses can classify amylases.

Biochemical Studies

Biochemical analysis of amylase genomic clones can be used to determineif any have a pH optima of less than pH 6. Lysates of these genomicclones can be tested for thermal tolerance by incubation at 70° C., 80°C., 90° C. or 100° C. for 10 minutes and measurement of residualactivity at pH 4.5. Those clones retaining >50% activity after heattreatment at 80° C. are chosen for further analysis. These clones can beincubated at 90° C. for 10 minutes at pH 6.0 and 4.5 and tested forresidual activity at pH 4.5. Thermal activity of the clones withresidual activity after heat treatment at 90° C. at pH 4.5 can bemeasured at room temperature, 70° C. and 90° C. at pH 4.5.

In one aspect, starch-degrading enzymes are screened for activity on rawstarch and ‘resistant’ starch for any form of hydrolase activity,including amylase, pullulanase, cyclodextrin glycosyltransferase,glucoamylases other any other glucosidase activity. In one aspect, theidentified active enzymes are characterized, e.g., by specific activityand specificity for branched sugars and/or longer oligosaccharides.

In one aspect, fungal isolates are investigated for novel enzymes using,e.g., probes and/or discovery processes of the invention, for example,screening gDNA and/or cDNA libraries made from animals, microorganismsor insects, e.g., the gut contents of insects that attack and consumestored grains, including screening environmental libraries forunder-represented enzymes. Combinations of enzymes can be evaluated.

In one aspect, stability tests are performed: e.g., purified enzymes intheir own storage buffer, as compared to activity of fresh enzyme, iftested; e.g., aliquots of 20 ul stored at lower temperatures, e.g., atbetween about 4° C. to −20° C. to −80° C.; and in one aspect, activityis retested in granular and soluble starch monthly

Amylases can be evaluated under a variety of conditions. In thefollowing protocols No 2 yellow dent corn can be used as a starchsource.

Exemplary Liquefaction Assay

A starch slurry comprising 35% dry solids (“DS”) is subjected to primaryliquefaction for five minutes under various temperatures in the range of95° C. to 119° C. (e.g., at about 110° C.), with an enzyme concentrationof between 0.2 to 0.8 gram/kilogram (g/kg) starch DS, with added calciumin the range of between zero and 30 parts per million (ppm), at pH 4.0to pH 5.6. Secondary liquefaction comprised conditions of 120 minutes at95° C.

Exemplary Saccharification Assay

Saccharification is initially tested using 35% dry solids (“DS”) (starchslurry) and glucoamylase AMG 300L (Novozymes A/S, Denmark) at 0.225AGU/gram DS (AGU=amyloglucosidase, or glucoamylase, units), pH 4.3, at60° C. for 44 hours.

In one aspect, exemplary amylases and/or glucoamylases of the inventionare used in a dosage range of between 0.5 to 0.7 kg/MT DS starch.

The invention provides methods for making nutritive sweeteners usingenzymes of the invention, e.g., processes comprising the above describedliquefaction and saccharification protocols using any amylase and/orglucoamylase of the invention. In one aspect, the dosage range for anenzyme of the invention in these processes is between about 0.5 to 0.7gram per kg starch DS, a jet temperature (e.g., using a jet cooker) ofabout 110° C., pH 4.5, no added calcium.

Dry Mill Ethanol Production

The invention provides methods for Dry Mill Ethanol Production usingenzymes of the invention. In evaluating enzymes of the invention for usein Dry Mill Ethanol Production, particularly, liquefaction of dry millcorn flour, a bench scale reactor can be used with corn flour sourcedfrom commercial dry mill. TERMAMYL™ SC (Novozymes A/S, Denmark) amylasecan be used as a competitive benchmark. In alternative aspects, optimumconditions are 85° C., pH 5.7. Five independent variables can bestudied: temperature (in a range of between 80° C. to 100° C.), enzymedose of between 0.2 to 1.0 g/kg starch, pH 4.4 to 6.0, calcium in arange between 0 ppm to 200 ppm, and a recycled backset between about 0%to 40%.

At 95° C., in some embodiments, amylases and/or glucoamylases of theinvention can reduce viscosity of dry mill corn flour more rapidly thanTERMAMYL™ SC (Novozymes A/S, Denmark) amylase at its optimum conditions,including at 85° C. The rate of viscosity reduction by amylases can beinfluenced most by enzyme dose and temperature. Alternative optimalranges can be in the range of 0.4 to 0.6 g/kg starch, with an optimumtemperature at 95° C.

In some embodiments, amylases and/or glucoamylases of the invention canbe effective at a lower pH and a higher temperature than TERMAMYL™ SC(Novozymes A/S, Denmark) amylase at a pH in the range between pH 4.4 andpH 5.6. Calcium addition can have a minimal effect on rate of viscosityreduction at 95° C.

The activities of the purified enzymes were compared in differentstorage buffers, as listed below, after 1 week of incubation at 37° C.The buffer with the lowest loss of activity compared to the activity ofthe same enzyme kept at +4° C. was selected as the storage buffer ofchoice. Exemplary assay conditions to test for amylase activity, e.g.,to determine if a polypeptide of the invention retains activity underparticular conditions, include (MPB: Methylparaben):

-   -   20% Glucose, 0.1% MPB in PBS, pH 7.2@37° C.    -   20% Glucose, 0.1% MPB in Acetate Buffer, pH 5@37° C.    -   20% Sucrose, 0.1% MPB in PBS, pH 7.2@37° C.    -   20% Sucrose, 0.1% MPB in Acetate Buffer, pH 5@37° C.    -   0.1% MPB in PBS, pH 7.2@37° C.    -   0.1% MPB in Acetate Buffer, pH 5@37° C.    -   PBS, pH 7.2@37° C.    -   Acetate Buffer, pH 5@37° C.    -   20% Glucose, 0.1% MPB in PBS, pH 7.2@4° C.    -   20% Glucose, 0.1% MPB in Acetate Buffer, pH 5@4° C.    -   20% Sucrose, 0.1% MPB in PBS, pH 7.2@4° C.    -   20% Sucrose, 0.1% MPB in Acetate Buffer, pH 5@4° C.    -   20% Maltose in PBS (50 mM sodium phosphate pH7.5; 100 mM NaCl)    -   0.1% MPB in PBS, pH 7.2@4° C.    -   0.1% MPB in Acetate Buffer, pH 5@4° C.    -   PBS, pH 7.2@4° C.    -   Acetate Buffer, pH 5@4° C.

Exemplary enzymes of the invention that have been tested under theseexemplary assay conditions include: SEQ ID NO:26; SEQ ID NO:18; SEQ IDNO:4; SEQ ID NO:28; SEQ ID NO:48; SEQ ID NO:76; SEQ ID NO:52; SEQ IDNO:70; SEQ ID NO:66. For SEQ ID NO:26 and SEQ ID NO:4, 20% sucrose in1×PBS, pH 7 with 0.1% methyl paraben (SMP*) was chosen, and for SEQ IDNO:18, 20% sucrose in 50 mM sodium acetate with 150 mM sodium chlorideand 0.1% methyl paraben (SMA**) was chosen. FIG. 13 illustrates aninitial rate comparison (using Vmax) of the exemplary enzyme SEQ IDNO:70 in different storage buffers (as indicated in the figure); 0.15 ugtotal protein per reaction; FIG. 14 illustrates an initial ratecomparison (using Vmax) of the exemplary enzyme SEQ ID NO:76 indifferent storage buffers (as indicated in the figure); 0.5 ug totalprotein per reaction; FIG. 15 illustrates an initial rate comparison(using Vmax) of the exemplary enzyme SEQ ID NO:4 in different storagebuffers (as indicated in the figure); FIG. 16 illustrates an initialrate comparison (using Vmax) of the exemplary enzyme SEQ ID NO:66 indifferent storage buffers (as indicated in the figure); 0.03 ug totalprotein per reaction; FIG. 17 illustrates an initial rate comparison(using Vmax) of the exemplary enzyme SEQ ID NO:52 in different storagebuffers (as indicated in the figure); 0.025 ug total protein perreaction; FIG. 18 illustrates an initial rate comparison (using nmole ofglucose per min per ug of enzyme) of the exemplary enzyme SEQ ID NO:28in different storage buffers (as indicated in the figure); FIG. 19illustrates an initial rate comparison (using nmole of glucose per minper ug of enzyme) of the exemplary enzyme SEQ ID NO:26 in differentstorage buffers (as indicated in the figure); FIG. 20 illustrates aninitial rate comparison (using nmole of glucose per min per ug ofenzyme) of the exemplary enzyme SEQ ID NO:18 in different storagebuffers (as indicated in the figure).

Bradford and BCA assays as well as SDS PAGE and Absorbance at A280 wereused to determine the concentration of the purified proteins. Thestandardized amylase solution (Sigma A6211) was used as a reference inthese assays, and BSA. Concentration by BCA and A280 was similar, butnot by Bradford using BSA as a standard.

In these assays, protein (enzyme) was purified by precipitation;hydrophobic interaction; size exclusion; ion exchange; affinity; and inone exemplary protocol, protein (enzyme) was purified by precipitation,hydrophobic interaction, ion exchange and as a last step, affinity(chromatography).

The exemplary SEQ ID NO:18, SEQ ID NO:4 and SEQ ID NO:26 were purifiedby either ammonium sulfate (SEQ ID NO:26) or ethanol precipitation (SEQID NO:18 and SEQ ID NO:4). Lyophilized supernatants from culturesexpressing the enzymes of interest were resuspended in water at aconcentration of about 1 g/5 ml. SEQ ID NO:18 and SEQ ID NO:4 wereprecipitated by addition of cold ethanol (2 volumes of proteinsolution). SEQ ID NO:26 was precipitated in 80% ammonium sulfatesolution. After precipitation the protein suspensions were dialyzed onceagainst water and subsequently against storage buffer.

Stability of purified exemplary enzymes of this invention was tested ineither PBS at pH 7 or PBS pH 7.5 (in this case, the parameters were 50mM sodium phosphate; 100 mM NaCl) or in 50 mM sodium acetate pH 5.2 with150 mM NaCl added. Glucose (20%) or sucrose (20%) was added to thebuffers as well as an antimicrobial, 0.1% methyl paraben (Sigma). Theenzymes were incubated in their respective buffers at 37° C. for 1 weekand the enzyme activity was tested and compared to the activity of thesame enzyme stored at +4° C.

The concentration of each purified enzyme was estimated using 4different methods: Bradford assay with amylase Sigma A-6211 as standard,BCA assay with Sigma A-6211 as standard, absorbance at 280 nm in 8Murea, and densitometric measurement of stained protein after SDS PAGE,using Sigma A-6211 amylase as the reference. The numbers obtained withthese different methods for each of the purified proteins are presentedin Table 3.1. For final quantification, the concentration obtained bymeasuring absorbance at 280 nm in 8M urea was used.

A summary of the large scale purification for these assays is:

Summary of Recover Amount based on Absorbance Value mg/ml Volume (ml)Total mg SEQ ID NO: 18 53.94 111 5987.34 SEQ ID NO: 26 39.23 86.83405.164 SEQ ID NO: 4 70.01 55.4 3878.554

Summary of Purity Before Purification After Purification A260/Expression A260/ Expression A280 Ratio A280 Ratio SEQ ID NO: 18 0.52 91%0.59 92% SEQ ID NO: 26 0.56 85% 0.65 86% SEQ ID NO: 4 0.67 91% 0.73 85%

Summary of Activity Raw Starch Soluble Purified Unpurified PurifiedUnpurified SEQ ID NO: 18 10.7228 ± 0.7925  6.4828 ± 0.5681 40.6725 ±1.7016 24.7354 ± 3.821 SEQ ID NO: 26 5.7837 ± 0.2271 4.0691 ± 0.286417.4833 ± 0.7831  14.1849 ± 0.3761 SEQ ID NO: 4 6.47 ± 0.34 4.074 ±0.35  127.37 ± 1.78  116.52 ± 1.27

Estimation of Enzyme Concentration by Different Methods AbsorbanceBradford BCA (A6211) (Urea) SEQ ID NO: 18 71.10 57.64 53.94 SEQ ID NO:26 43.98 57.64 39.29 SEQ ID NO: 4 69.20 57.64 70.01

Example 2: Thermostable Amylases Active at Alkaline pH

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention, e.g., is athermostable amylase.

Commercial automatic dish wash (ADW) formulations can be used todetermine if a polypeptide is within the scope of the invention, e.g.,is a thermostable amylase. Studies can include the identification ofhigh pH amylases; and enzymes having the ability to degrade starch. DNAsequence and bioinformatics analyses can classify many of these genes asamylases, or having other enzyme specificities, e.g., neopullulanases,amylopullulanases and amylomaltases.

Biochemical Studies

One exemplary method for determining if a polypeptide is within thescope of the invention, e.g., is a thermostable amylase, is to test foractivity where the enzyme can hydrolyze starch at alkaline pH, e.g., upto pH 10, and about 50° C.

Soluble protein is purified to homogeneity and specific activity(units/mg, where 1 unit=μmol reducing sugars/min) is measured at pH 8and pH 10 (40° C. and 50° C.) using 2% starch in buffer. Specificactivity can be determined by removing samples at various time pointsduring a 30 minute reaction and analyzing for reducing sugars. Theinitial rate can be determined by fitting the progress curves to alinear equation.

Stability Studies

Stability in the presence of the ADW formulation can be measured bybiochemical analysis. The benchmark for these studies can be acommercial enzyme in the formulation matrix. The measured activity afterthe incubation can be expressed as a percentage of the originalactivity.

Wash Tests

Wash tests using starch coated slides can be carried out to gauge theperformance of each a purified enzyme of the invention as compared to acommercial amylase. Spaghetti starch coated slides can be preparedaccording to protocol: two pre-weighed starch coated slides are placedback to back in a 50 mL conical tube and 25 mL of ADW solution, +/−enzyme are added per tube. The tubes are incubated for 20 minutes at 50°C. with gentle rotation on a vertical carousel. Following the incubationperiod, the slides are immediately rinsed in water and oven driedovernight. All trials are run in duplicate and the commercial enzyme wasrun as a positive control. The results can be expressed as net % starchremoved, e.g. % of starch removed in ADW with enzyme, minus the % ofstarch removed in ADW alone.

Example 3: Gene Optimization

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention, e.g., assessing enzymeperformance in the presence of ADW performance.

The properties of enzymes may be improved by various evolutionstrategies, including Gene Site Saturation Mutagenesis™ (GSSM™) andGeneReassembly™ technologies (Diversa Corporation, San Diego, Calif.).Such techniques will be applied to the amylase nucleic acids of theinvention in order to generate pools of variants that can be screenedfor improved performance. In one aspect, parental molecules forevolution include any nucleic acid of the invention, e.g., sequencesencoding SEQ ID NO:2; SEQ ID NO:4; SEQ ID NO:6, etc.

A high throughput screen (HTS) can be used to assess enzyme performancein the presence of ADW performance. HTS can be automated and has showedconsistent results for the parental amylases.

Example 4: Characterization of α-Amylases Having Activity at Alkaline pH

The following example describes exemplary methods for determining if apolypeptide is within the scope of the invention, for example, hasalpha-amylase activity at alkaline pH.

Amylases and/or glucoamylases of the invention having activity atalkaline pH can be characterized using kinetics on 2% starch at pH 8 and10 (40° C. and 50° C.). 1 unit of activity can be defined as release of1 μmol reducing sugars per minute.

Example 5: Amylase Activity Assay: BCA Reducing Ends Assay

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention, for example, by a BCAreducing ends assay. Amylase activity of clones of interest can bedetermined using the following methodology.

-   -   1. Prepare 2 substrate solutions, as follows:    -   a) 2% soluble starch (potato or granular corn starch) pH 8        solution by dissolving 2 gm potato starch in 100 ml 100 mM        sodium phosphate pH 8).    -   b) 2% soluble starch (potato) pH 10 solution by dissolving 2 gm        potato starch in 100 ml 100 mM sodium carbonate.

Heat both solutions in a boiling water bath, while mixing, for 30-40minutes until starch dissolves.

-   -   2. Prepare Solution A from 64 mg/ml sodium carbonate        monohydrate, 24 mg/ml sodium bicarbonate and 1.95 mg/ml BCA        (4,4′-dicarboxy-2,2′-biquinoline disodium salt (Sigma Chemical        cat # D-8284). Added above to dH2O.    -   3. Prepare solution B by combining 1.24 mg/ml cupric sulfate        pentahydrate and 1.26 mg/ml L-serine. Add mixture to dH2O.    -   4. Prepare a working reagent of a 1:1 ration of solutions A and        B.    -   5. Prepare a Maltose standard solution of 10 mM Maltose in dH2O,        where the 10 mM maltose is combined in 2% soluble starch at        desired pH to a final concentration of 0, 100, 200, 300, 400,        600 μM. The standard curve will be generated for each set of        time-points. Since the curve is determined by adding 10 ul of        the standards to the working reagent it works out to 0, 1, 2, 3,        4, 6 nmole maltose.    -   6. Aliquot 1 ml of substrate solution into microcentrifuge        tubes, equilibrate to desired temperature (5 min) in heat block        or heated water bath. Add 50 ul of enzyme solution to the inside        of the tube lid.    -   7. While solution is equilibrating mix 5 ml of both solution A        & B. Aliquot 100 ul to 96 well PCR plate. Set plate on ice.    -   8. After 5 minute temperature equilibration, close lid on tubes,        invert and vortex 3 times. Immediately aliquot 10 ul into plate        as t=0 (zero time point). Leave enzyme mixture in heat block and        aliquot 10 ul at each desired time point (e.g. 0, 5, 10, 15, 20,        30 minutes).    -   9. Ensure that 12 wells are left empty (only working reagent        aliquotted) for the addition of 10 ul of standards, for the        standard curve.    -   10. When all time points are collected and standards are added,        cover plate and heated to 80° C. for 35 min. Cool plate on ice        for 10 min. Add 100 ul H2O to all wells. Mix and aliquot 100 ul        into flat bottomed 96-well plate and read absorbance at 560 nm.    -   11. Zero each sample's time points against its own t=0 (subtract        the average t=0 A560 value from other average A560 values).        Convert the A560_((experimental)) to umole (Divide        A560_((experimental)) by the slope of the standard curve        (A560/umole).        Generate a slope of the time points and the umole (in        umole/min), multiply by 100 (as the umole value only accounts        for the 10 ul used in the assay, not the amount made in the 1 ml        rxn). To get the specific activity divide the slope (in        umole/min) by the mg of protein. All points should be done at a        minimum in duplicate with three being best.

Divide protein concentration (mg/ml) by any dilution to get mg used inassay.

Divide the above slope by mg used in assay to get specific activity

Specific Activity=24.93 umole/min/mg

See for example, Dominic W. S. Wong, Sarah B. Batt, and George H.Robertson (2000) J. Agric. Food Chem. 48:4540-4543; Jeffrey D. Fox andJohn F. Robyt, (1991) Anal. Biochem. 195, 93-96.

Example 6: Screening for α-Amylase Activity

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention. Amylase activity ofclones can be assessed by a number of methods known in the art. Thefollowing is an exemplary methodology. The number of plaques screened,per plate, can be approximately 10,000 pfu's. For each DNA library: atleast 50,000 plaques per isolated library and 200,000 plaques pernon-isolated library should be screened depending upon the pfu titer forthe λ Zap Express amplified lysate.

Titer Determination of Lambda Library

-   1) μL of Lambda Zap Express amplified library stock added to 600    μL E. coli MRF′ cells (OD₆₀₀=1.0). To dilute MRF′ stock, 10 mM MgSO₄    is used.-   2) Incubate at 37° C. for 15 minutes.-   3) Transfer suspension to 5-6 mL of NZY top agar at 50° C. and    gently mix.-   4) Immediately pour agar solution onto large (150 mm) NZY media    plate.-   5) Allow top agar to solidify completely (approximately 30 minutes),    then invert plate.-   6) Incubate the plate at 39° C. for 8-12 hours.-   7) Number of plaques is approximated. Phage titer determined to give    10,000 pfu/plate. Dilute an aliquot of Library phage with SM buffer    if needed.

Substrate Screening

-   1) Lambda Zap Express (50,000 pfu) from amplified library added to    600 μL of E. coli MRF′ cells (OD₆₀₀=1.0). For non-environment    libraries, prepare 4 tubes (50,000 pfu per tube).-   2) Incubate at 37° C. for 15 minutes.-   3) While phage/cell suspensions are incubating, 1.0 mL of red starch    substrate (1.2% w/v) is added to 6.0 mL NZY top agar at 50° C. and    mixed thoroughly. Keep solution at 50° C. until needed.-   4) Transfer ⅕ (10,000 pfu) of the cell suspension to substrate/top    agar solution and gently mixed.-   5) Solution is immediately poured onto large (150 mm) NZY media    plate.-   6) Allow top agar to solidify completely (approximately 30 minutes),    then invert plate.-   7) Repeat procedures 4-6 4 times for the rest of the cell suspension    (⅕ of the suspension each time).-   8) Incubate plates at 39° C. for 8-12 hours.-   9) Plate observed for clearing zones (halos) around plaques.-   10) Plaques with halos are cored out of agar and transferred to a    sterile micro tube. A large bore 200 μL pipette tip works well to    remove (core) the agar plug containing the desired plaque.-   11) Phages are re-suspended in 500 μL SM buffer. 20 μL Chloroform is    added to inhibit any further cell growth.-   12) Pure phage suspension is incubated at room temperature for 4    hours or overnight before next step.

Isolation of Pure Clones

-   1) 10 μL of re-suspended phage suspension is added to 500 μL of E.    coli MRF′ cells (OD₆₀₀=1.0).-   2) Incubate at 37° C. for 15 minutes.-   3) While phage/cell suspension is incubating, 1 mL of red starch    substrate (1.2% w/v) is added to 6.0 mL NZY top agar at 50° C. and    mixed thoroughly. Keep solution at 50° C. until needed.-   4) Cell suspension is transferred to substrate/top agar solution and    gently mixed.-   5) Solution is immediately poured onto large (150 mm) NZY media    plate.-   6) Allow top agar to solidify completely (approximately 30 minutes),    then invert plate.-   7) Plate incubated at 39° C. for 8-12 hours.-   8) Plate observed for a clearing zone (halo) around a single plaque    (pure clone). If a single plaque cannot be isolated, adjust titer    and re-plate phage suspension.-   9) Single plaque with halo is cored out of agar and transferred to a    sterile micro tube. A large bore 200 μL pipette tip works well to    remove (core) the agar plug containing the desired plaque. To    amplify the titer, core 5 single active plaques into a micro tube.-   10) Phages are re-suspended in 500 μL SM buffer. 20 μL Chloroform is    added to inhibit any further cell growth.-   11) Pure phage suspension is incubated at room temperature for 4    hours or overnight before next step. The pure phage suspension is    stored at −80° C. by adding DMSO into the phage suspension (7% v/v).

Excision of Pure Clone

-   1) 100 μL of pure phage suspension is added to 200 μL E. coli MRF′    cells (OD600=1.0). To this, 1.0 μL of EXASSIST helper phage (>1×106    pfu/mL; Stratagene) is added. Use 2059 Falcon tube for excision.-   2) Suspension is incubated at 37° C. for 15 minutes.-   3) 3.0 mL of 2×YT media is added to cell suspension.-   4) Incubate at 30° C. for at least 6 hours or overnight while    shaking.-   5) Tube transferred to 70° C. for 20 minutes. The phagemid    suspension can be stored at 4° C. for 1 to 2 months.-   6) 100 μL of phagemid suspension transferred to a micro tube    containing 200 μL of E. coli Exp 505 cells (OD600=1.0).-   7) Suspension incubated at 37° C. for 15 minutes.-   8) 300 μL of SOB is added to the suspension.-   9) Suspension is incubated at 37° C. for 30 to 45 minutes.-   10) 100 μL of suspension is transferred to a small (90 mm) LB media    plate containing Kanamycin (LB media with Kanamycin 50 μg/mL) for    Zap Express DNA libraries or Ampicillin (LB media with Kanamycin 100    μg/mL) for Zap II DNA libraries.-   11) The rest of suspension is transferred to another small LB media    plate.-   12) Use sterile glass beads to evenly distribute suspension on the    plate.-   13) Plates are incubated at 30° C. for 12 to 24 hours.-   14) Plate observed for colonies.-   15) Inoculate single colony into LB liquid media containing suitable    antibiotic and incubate at 30° C. for 12 to 24 hours.-   16) Glycerol stock can be prepared by adding 80% glycerol into    liquid culture (15% v/v) and stored at −80° C.

Activity Verification

-   1) 50 μL of liquid culture is transferred to a micro tube. Add 500    μL of 8% pH7 Amylopectin Azure into the same tube. Prepare 2 tubes    for each clone.-   2) Activity is tested at 50° C. for 3 hours and overnight. Use pH 7    buffer as control.-   3) Cool the test specimen at ice-water bath for 5 minutes.-   4) Add 750 μL of Ethanol and mixed thoroughly.-   5) Centrifuge at 13000 rpm (16000 g's) for 5 minutes.-   6) Measure OD of the supernatant at 595 nm.

RFLP Analysis

-   1) 1.0 mL of liquid culture is transferred to a sterile micro tube.-   2) Centrifuge at 13200 rpm (16000 g's) for 1 minute.-   3) Discard the supernatant. Add another 1.0 mL of liquid culture    into the same sterile micro tube.-   4) Centrifuge at 13200 rpm (16000 g's) for 1 minute.-   5) Discard the supernatant.-   6) Follow QIAPREP™ spin mini kit protocol for plasmid isolation.-   7) Check DNA concentration using BioPhotometer.-   8) Use Sac I and Kpn I for first double digestion. Incubate at    37° C. for 1 hour.-   9) Use Pst I and Xho I for second double digestion. Incubate at    37° C. for 1 hour.-   10) Add Loading dye into the digested sample.-   11) Run the digested sample on a 1.0% agarose gel for 1-1.5 hours at    120 volts.-   12) View gel with gel imager. All clones with a different digest    pattern can be sequence analyzed.

Example 7: Assay for Amylases

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention.

Preparation of Host Cultures

-   -   1. Start an overnight culture of XL1-BLUE™ MRF′ host cells. Use        a single colony from a streak plate to inoculate 10 mL LB        supplemented with 20 ug/mL tetracycline. Grow overnight culture        shaking at 37° C. for at least 16 hours.    -   2. Using aseptic technique, inoculate a fresh 100 mL of LB_(Tet)        day culture with XL1-BLUE™ MRF′ host from the overnight LB Tet        culture.    -   3. Grow in a 37° C. shaker until the OD reaches 0.75-1.0.    -   4. Pellet host cells at 1000×g for 10 minutes and gently        resuspend in 10 mM MgSO₄ at OD5.    -   5. Dilute a small amount of host cells to OD1 for use in        titering and pintooling.    -   6. Host preparations can be used for up to 1 week when stored on        ice or at 4° C.        -   To shorten growth time for the day culture, use ½× the usual            Tet concentration in LB (½X=10 ug/mL), or omit the            antibiotic altogether.        -   Do not use NZY when selecting with Tetracycline. The high            Mg⁺⁺ concentration in NZY medium renders Tet inactive.

Titering Lambda Libraries

-   -   7. Place three sterile microfuge tubes in a rack.    -   8. Aliquot 995 uL prepared host cells in one tube and 45 uL        prepared OD1 host cells into each of the two remaining tubes.    -   9. Add 5 uL of lambda library to the tube containing 995 uL host        cells and mix by vortexing. This results in a dilution factor of        200.    -   10. Prepare 1/2,000 and 1/20,000 dilutions by consecutively        adding 5 uL of previous dilution to the remaining two tubes        containing 45 uL prepared host cells. Mix by vortexing after        each dilution was made.    -   11. Allow phage to adsorb to host by incubating at 37° C. for 15        minutes.    -   12. Meanwhile, pipet 100 uL of prepared OD1 host cells to each        of three Falcon 2059 tubes.    -   13. Add 5 uL of each dilution to a separate 2059 tube containing        host cells.    -   14. Plate each by adding 3 mL top agar to each tube and quickly        pour over 90 mm NZY plates. Ensure a smooth, even distribution        before the top agar hardens.    -   15. Invert plates and incubate at 37° C. overnight.    -   16. Count plaques and calculate titer of the library stock (in        plaque forming units (pfu) per uL).

Lambda Microtiter Screening for Amylases

Preparation

-   -   1. Prepare a sufficient amount of XL1-Blue MRF′ host culture, as        described above, for the amount of screening planned. A culture        of 100 mL is usually sufficient for screening 2-3 libraries.    -   2. Autoclave several bottles compatible with the QFill2        dispenser. These are the wide-mouth Corning bottles, 250 mL        containing a sealing ring around the lip.    -   3. Make sure there are sufficient amounts of plates, top agar,        BODIPY starch, red starch solution, etc. available for the        screen.    -   4. Schedule the Day 2 robot run with a representative from        Automation.

Day 1

-   -   1. Label the 1536-well plates (black) with library screen and        plate number. Tough-Tags™ tube stickers, cut in half width-wise,        are ideal for labeling 1536 well plates.    -   2. Calculate volumes of library, host cells and NZY medium        necessary for the screen. This is easily done with an Excel        spreadsheet.    -   3. Combine the calculated volumes of lambda library and OD5 host        cells in a sterile 250 mL wide-mouth Corning bottle (containing        a sealing ring).    -   4. Allow adsorption to occur at 37° C. for 15 minutes.    -   5. Add the calculated volume of NZY medium and mix well. This is        referred to as the cell-phage-medium suspension.    -   6. Perform a concomitant titer by combining 50 uL of the        cell-phage-medium suspension with 250 uL of OD1 host cells in a        Falcon 2059 tube, then plating with 9 mL of top agar onto a 150        mm NZY plate. Incubate concomitant titer plate at 37° C.        overnight.    -   7. Load the dispenser with the remainder of the suspension and        array each labeled 1536-well plate at 4 uL per well. If the        dispenser leaves air bubbles in some wells, they can be removed        by centrifuging the plates at 200×g for 1 minute.    -   8. Add 0.5 uL of positive control phage to well position AD46 of        at least two of the assay plates. Use a strong amylase-positive        lambda clone for this purpose.    -   9. Incubate assay plates at 37° C. overnight in a humidified        (≥95%) incubator.

Day 2

-   -   1. Count the pfu on the concomitant titer plate and determine        the average seed density per well (in pfu per well).    -   2. Pintool at least 2 plates of each library screen (preferably        the 2 containing positive controls) as follows:        -   a) Prepare 2 host lawn plates to act as a surface on which            to pintool: combine 250 uL of OD1 host cells with 2 mL 2%            red starch and plate with 9 mL top agar onto 150 mm NZY            plates. Hold each plate as level as possible as the top agar            solidifies in order to produce an even hue of red across the            plate.        -   b) Using a twice flame-sterilized 1536 position pintool,            replicate at least 2 of the screening plates onto the host            lawn plates.        -   c) Place the pintooled recipient plates in a laminar flow            hood with the lids off for about 15-30 minutes (to vent off            excess moisture).        -   d) Replace the lids and incubate inverted at 37° C.            overnight.    -   3. Prepare the 2×BODIPY starch substrate buffer as follows:        -   a) Calculate the total volume of 2× substrate buffer            solution needed for all screening plates at 4 uL per well            (including any extra deadspace volume required by the            dispenser) and measure this amount of 100 mM CAPS pH 10.4            into a vessel appropriate for the dispenser used.        -   b) Retrieve enough 0.5 mg tubes of BODIPY starch to produce            the required volume of 2× substrate buffer [calculated in            step a) above] at a final concentration of 20-30 ug/mL.        -   c) Dissolve each 0.5 mg tube in 50 uL DMSO at room            temperature, protected from light, with frequent vortexing.            This takes more than 15 minutes; some production lots of            BODIPY starch dissolve better than others.        -   d) Add 50 uL 100 mM CAPS buffer pH 10.4 to each tube and mix            by vortexing.        -   e) Pool the contents of all tubes and remove any undissolved            aggregates by centrifuging for 1 minute at maximum speed in            a microfuge.        -   f) Add the supernatant to the rest of the 100 mM CAPS buffer            measured in step a) above.        -   g) Protect the 2× substrate buffer from light by wrapping in            foil.    -   4. Take plates and substrate buffer to the automation room and        program the robot with the following parameters:        -   a) dispense 4 uL substrate buffer per well        -   b) 1^(st) read at 1 hour post-substrate, 2^(nd) read at 9            hours, and third read at 17 hours; with 37° C. incubation            between reads        -   c) excitation filter: 485 nm; emission filter: 535 nm        -   d) set the Spectrafluor gain at 70, or the optimal gain for            the batch of 2× substrate buffer prepared.        -   e) ensure that the incubator used will protect assay plates            from light.

Day 3

-   -   1. Check pintooled plates for clearings in the bacterial lawn at        all positions corresponding to wells on the associated assay        plate. Also check for clearings in the red starch in any of the        pin positions. If plates containing positive controls were used        for pintooling, you should be able to see a large clearing zone        in the red background. Be wary of contaminants that also form        clearing zones in red starch (see comment “Contaminants That        Form Clearing Zones in Red Starch” at end of Example 7).    -   2. Identify putative hits from the data file produced by the        robot computer. The KANAL program produced by Engineering        simplifies data analysis. As a rule of thumb, a putative hit is        characterized as a well having signal intensity rising at least        1.5 fold over background.    -   3. For each putative, remove 2 uL from the well and add to a        tube containing 500 uL SM buffer and 50 uL CHCl3. Vortex to mix        and store at 4° C. This solution will be referred to hereafter        as the 4e-3 stock. The original screening plates should be        stored at 4° C., protected from light, at least until breakouts        are complete.

This is an exemplary method of breaking out putative hits. It is aliquid phase assay that relies on confirmation of activity on BODIPYstarch. Alternatively, putative hits can be plated directly onto solidphase plates containing red starch such that 2,000-3,000 pfu per hit areexamined for clearing zones. However, inability to observe clearingzones on red starch is not necessarily an indication that a putative hitwas a false positive. It would then need to be assayed using the formatin which it was originally identified (i.e., liquid phase using BODIPYstarch as substrate). In addition, very weak positives are more easilyidentified using the method detailed below.

Day 1

-   -   1. In a sterile 50 mL conical tube, combine 0.5 mL OD5 host        cells with 45.5 mL NZY. This will be referred to as the        host-medium suspension.    -   2. For each putative hit to be analyzed, aliquot 1 mL of        host-medium suspension into each of 3 three sterile microfuge        tubes.    -   3. Set the 12-channel pipetman in multidispense mode with an        aliquot size of 20 uL and an aliquot number of 2×. Mount the        pipetman with a clean set of sterile tips.    -   4. Pour about 1 mL of host-medium suspension into a new sterile        solution basin and load the multichannel pipetman.    -   5. Dispense 20 uL per well into the last row (row P) of a black        384-well plate (12 channels×2=24 wells). This row will be used        later for the controls.    -   6. Expel the remaining liquid in the tips by touching the tips        against the surface of the basin and pressing the RESET button        on the pipetman. Lay the pipetman down in a way to prevent        contamination of the tips. There is no need to change the tips        at this point.    -   7. Pour the remainder of the fluid in the basin into a waste        container (like a beaker) taking care to avoid splash-back        contamination.    -   8. For the first putative to be analyzed, take 111 uL of the        4e-3 stock (see Day 2 in Lambda Microtiter Screening for        Amylases) and add it to the first in a set of three tubes        containing 1 mL host-medium suspension (step 2). Vortex to mix.        This is Dilution A.    -   9. Take 111 uL of Dilution A and add to the next tube in the        set. Vortex to mix. This is Dilution B.    -   10. Take 111 uL of Dilution B and add to the last tube in the        set. Vortex to mix. This is Dilution C. You should now have        three dilutions of phage, where concentrations of each differ by        a factor of 10.    -   11. Pour the contents of Dilution C (the most dilute of the 3        samples) into the solution basin and load the multichannel        pipetman.    -   12. Dispense 20 uL per well into the first row of the 384-well        plate (12 channels×2=24 wells).    -   13. Expel the remaining liquid in the tips by touching the tips        against the surface of the basin and pressing the RESET button        on the pipetman. Lay the pipetman down in a way to prevent        contamination of the tips. There is no need to change the tips        at this point.    -   14. Empty the basin as described above.    -   15. Pour the contents of Dilution B into the same basin and load        the multichannel pipetman.    -   16. Dispense 20 uL per well into the second row of the 384-well        plate.    -   17. Perform steps 13-16 similarly to dispense Dilution A into        the third row of the plate.    -   18. After all three dilutions have been arrayed into the first 3        rows of the plate, discard all tips and the solution basin into        the biohazardous waste container.    -   19. Mount the pipetman with a clean set of sterile tips and open        a new sterile solution basin.    -   20. Repeat steps 8-19 for each remaining putative hit, using        remaining rows on the plate up to row O. Five putative hits can        be analyzed on one 384-well plate, with the last row (row P)        saved for the controls.    -   21. Add 0.5 uL of each control to a separate well. Use at least        2-3 separate controls, preferably covering a range of activity.    -   22. Incubate assay plates at 37° C. overnight in a humidified        (≥95%) incubator.

Day 2

-   -   1. Pintool all breakout plates onto a host lawn with red starch        using the same method described for Day 2 in Lambda Microtiter        Screening for Amylases, except that a 384 position pintool is        used.    -   2. Prepare the 2×BODIPY starch substrate buffer as follows:        -   a) Calculate the total volume of 2× substrate buffer            solution needed for all breakout plates at 20 uL per well            (including any extra deadspace volume required by the            dispenser) and measure this amount of 100 mM CAPS pH 10.4            into a vessel appropriate for the dispenser used.        -   b) Retrieve enough 0.5 mg tubes of BODIPY starch to produce            the required volume of 2× substrate buffer [calculated in            step a) above] at a final concentration of 20-30 ug/mL.        -   c) Dissolve each 0.5 mg tube in 50 uL DMSO at room            temperature, protected from light, with frequent vortexing.            This takes more than 15 minutes; some production lots of            BODIPY starch dissolve better than others.        -   d) Add 50 uL 100 mM CAPS buffer pH 10.4 to each tube and mix            by vortexing.        -   e) Pool the contents of all tubes and remove any undissolved            aggregates by centrifuging for 1 minute at maximum speed in            a microfuge.        -   f) Add the supernatant to the rest of the 100 mM CAPS buffer            measured in step a) above.        -   g) Protect the 2× substrate buffer from light by wrapping in            foil.    -   3. Dispense 20 uL per well into all breakout plates.    -   4. Wrap all plates in aluminum foil and incubate at room        temperature for 2-6 hours.    -   5. Read each plate in the Spectrafluor with the following        settings:        -   a) fluorescence read (excitation filter: 485 nm; emission            filter: 535 nm)        -   b) plate definition: 384 well black        -   c) read from the top        -   d) optimal gain        -   e) number of flashes: 3    -   6. On the resulting Excel spreadsheet, chart each putative's 3        rows in a separate graph and check for activity. Ensure that the        positives controls produced signals over background.    -   7. For each putative that appears to have a real signal among        the wells, harvest a sample from a positive well as follows:        -   a) Select a positive well from a row representing the            highest initial dilution.        -   b) Transfer 2 uL from that well into a tube containing 500            uL SM and 50 uL CHCl₃. This is referred to as the breakout            stock.        -   c) Store at 4° C.    -   8. Using methods previously described, plate about 10 uL of each        breakout stock onto 150 mm NZY plates using red starch. The        objective is to obtain several (at least 20) well-separated        plaques from which to core isolates.

Day 3

-   -   1. Check pintooled plates for an acceptable incidence of        clearings in the bacterial lawn corresponding to wells on the        associated assay plate. Also check for clearings in the red        starch in the positive controls and in any tested putatives. Be        wary of contaminants that also form clearing zones in red starch        (see below).    -   2. From the solid phase plates containing dilutions of breakout        stocks, core several isolated plaques, each into 500 uL SM with        50 uL CHCl₃. This is referred to as the isolate stock.    -   3. The isolate stocks can then be individually tested on BODIPY        starch using methods described above. This step can be skipped        if the plaque that was cored in step 2 produced a clearing zone        in the red starch background. The isolate stocks were then be        individually tested on BODIPY starch using methods described        above. However, this step may be skipped if the plaque that was        cored in step 2 produced a clearing zone in the red starch        background.

Excisions

Day 1

-   -   1. In a Falcon 2059 tube, mix 200 uL OD1 XL1-Blue MRF′ host, 100        uL lambda isolate stock and 1 uL EXASSIST™ phage stock.    -   2. Incubate in 37° C. shaker for 15 minutes.    -   3. Add 3 mL NZY medium.    -   4. Incubate in 30° C. shaker overnight.

Day 2

-   -   1. Heat to excision tube to 70° C. for 20 minutes.    -   2. Centrifuge 1000×g for 10 minutes.    -   3. In a Falcon 2059 tube, combine 50 uL supernatant with 200 uL        EXP505™ OD1 host.    -   4. Incubate in 37° C. shaker for 15 minutes.    -   5. Add 300 uL SOB medium.    -   6. Incubate in 37 C shaker for 30-45 minutes.    -   7. Plate 50 uL on large LB_(Kan50) plate using sterile glass        beads. If the plates are “dry”, extra SOB medium can be added to        help disburse the cells.    -   8. Incubate plate at 30° C. for at least 24 hours.    -   9. Culture an isolate for sequencing and/or RFLP.

Growth at 30° C. reduces plasmid copy number and is used to mitigate theapparent toxicity of some amylase clones.

Contaminants that Form Clearing Zones in Red Starch

When using red starch on solid medium to assay phage for amylaseactivity, it is common to see contaminating colony forming units (cfu)that form clearing zones in the red starch. For pintooled plates, it isimportant to distinguish amylase-positive phage clones from thesecontaminants whenever they align with a particular well position. Thesource of the contaminating microbes is presumably the 2% red starchstock solution, which cannot be sterilized by autoclaving or byfiltering after preparation. It is thought that they are opportunisticorganisms that survive by metabolizing the red starch. In order toreduce these contaminants, sterile techniques can be used when making 2%red starch solutions and store the stocks either at 4° C. or on ice.

Example 8: Characterization of Alpha Amylase pH Optimum and SpecificActivity Determination

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention, e.g., by alpha amylaseactivity pH optimum and specific activity determination.

Enzymes of this invention can be used, for example, for both starchliquefaction for corn wet milling and desizing for textiles; e.g., insome embodiments enzymes of the invention have a pH optimum of 4.5 to5.0; at this lower pH, it is possible to use little or no calcium whichlowers overall operating costs and less byproduct formation. Inaddition, at this low pH, there is decreased chemical usage and ionexchange load. The industry standard B. licheniformis amylase issuboptimal in both thermostability and pH optimum. In some embodimentsenzymes of the invention have a higher application specific activitycompared to B. licheniformis amylase and therefore much less enzyme isrequired to hydrolyze a ton of starch (e.g., in some embodiments, asmuch as 20-fold less enzyme can be used).

The pH optimum for the hydrolysis of starch can be determined byreacting 50 uL of enzyme, 0.35 U/ml, with a 100 ml of 1% soluble starchsolution (0.0175 U/g of starch) for 30 minutes at 95 degrees C. Thereducing ends generated in the liquefied starch solution can be measuredby the neocupronine assay, described herein. The percent hydrolysis ofcornstarch can be determined by measuring the number of sugar reducingends produced with the neocupronine assay. Seventy grams of buffersolution (pH4-7) was weighed and 100 ppm of calcium can be added. Thirtygrams of cornstarch can be mixed into the buffer solution to form astarch slurry. The enzyme can be added and the vessels sealed andincubated at 95 degrees C. for 30 minutes with an initial heating rateof six degrees C. per minute. A 1 ml sample can be extracted from thereaction beakers and analyzed by the neocupronine assay. In someembodiments enzymes of the invention have an optimum between pH 4.5 and5, while the commercial B. licheniformis amylase performs optimally atabout pH 6.0.

Example 9: Amylase Activity Assays

The following example describes, inter alia, exemplary methods fordetermining if a polypeptide is within the scope of the invention, e.g.,by the assays described below.

Assay Using RBB-Starch

75 ul of RBB-starch substrate (1% RBB-insoluble corn starch in 50 mMNaAc buffer, pH=4.5) is added into each well of a new 96-well plate(V-bottom). Five micro-liters of enzyme lysate is transferred into eachwell with substrate using BIOMEK™ or ZYMARK™. The plates are sealed withaluminum sealing tape and shaken briefly on the shaker. The plates areincubated at 90° C. for 30 minutes, followed by cooling at roomtemperature for about 5 to 10 minutes. One hundred micro-liters of 100%ethanol is added to each well, the plates sealed and shaken briefly onthe shaker. The plates are then centrifuged 4000 rpm for 20 minutesusing bench-top centrifuge. 100 ul of the supernatant is transferredinto a new 96-well plate (flat bottom) by BIOMEK™ and read OD₅₉₅.Controls should be used.

Assay Using FITC-Starch

Add 50 ul of substrate (0.01% FITC-starch in 100 mM NaAc buffer, pH=4.5)into each well of a new 384-well plate. Transfer 5 ul of enzyme lysateinto each well with substrate and incubate the plate at room temperatureovernight. The polarization change of the substrate, excitation 485 nm,emission 535 nm, is read for each well. Controls should be used. 96 wellplates can be used for all assays.

Confirmation of New Active Clones

Each positive clone from screening is grown and induced using a standardprotocol. Each clone is examined for growth (i.e., cell density overtime), activity at per cell level (RBB-starch assay and liquefactionassay), expression (protein gel) and solubility of protein (bymicroscope analysis). The confirmed new elevated clones are transferredfor fermentation.

Example 10: Exemplary Protocol for Liquefying Starch and MeasuringResults

The following example described and exemplary protocol for liquefyingstarch using amylases and/or glucoamylases of the invention. Oneexemplary assay uses liquefied starch at pH 4.5 or 6.5 using thereaction conditions show below:

Reaction Conditions: 100 mM PO₄ pH 6.5, 1% (w/w) liquefied starch DE 12at 55° C. Both TLC and HPLC assays are done to verify activity. pHprofiles for the amylases to be tested are run using phosphate bufferpHed from 3.0-6.5, at 55° C. From the amount of observable hydrolysis,it can be visually demonstrated that some clones are more (or less)active at certain pH values than at other values at the above indicatedreaction conditions.

An exemplary protocol for the saccharification of liquefied starch at pH6.5:

-   -   Adjust the pH of the liquefied starch to the pH at which the        saccharification(s) is performed. Liquefy starch in 100 mM        sodium acetate buffer, pH 4.5 with 100 mM sodium phosphate salts        added so that before saccharification, the pH is adjusted to pH        6.5.    -   Weigh 5 gram samples of liquefied starch into tared bottles.    -   Use 0.04% (w/w) OPTIDEX L-400™ or approximately 400 mL of 1-10        diluted stock OPTIDEX L-400™ per 100 grams of liquefied starch.    -   Calculate the milligrams of OPTIDEX L-400™ contained in the 400        mL of 1-10 diluted stock OPTIDEX L-400™. Next, calculate the        volume of lysates needed to give the same concentration of        enzyme as the OPTIDEX L-400™.    -   Add enzymes to liquefied starch samples and incubate at desired        temperature (50 C°). After 18 hours determine DE and prepare a        sample for HPLC analysis.

An Exemplary DE Determination:

Exemplary Neocuproine Assay:

A 100 ml sample is added to 2.0 ml of neocuproine solution A (40 g/Lsodium carbonate, 16 g/L glycine, 0.45 g/L copper sulfate). To this isadded 2.0 ml of neocuproine solution B (1.2 g/L neocuproinehydrochloride-Sigma N-1626). The tubes are mixed and heated in a boilingwater bath for 12 minutes; cooled, diluted to 10 ml volume with DI waterand the OD read at 450 nm on the spectrophotometer. The glucoseequivalent in the sample is extrapolated from the response of a 0.2mg/ml glucose standard run simultaneously.

Exemplary HPLC Analysis:

Saccharification carbohydrate profiles are measured by HPLC (Bio-RadAminex HPX-87A column in silver form, 80° C.) using refractive indexdetection. Mobile phase is filtered Millipore water used at a flow rateof 0.7 ml/min. Saccharification samples are diluted 1-10 with acidifiedDI water (5 drops of 6 M HCl into 200 mL DI water) then filtered througha 0.45 mm syringe filter. Injection volume is 20 uL.

Exemplary TLC:

Reaction products were w/d at hourly timepoints and spotted and dried ona TLC plate. The Plate was then developed in 10:90 water:isopropanol andvisualized with either a vanillin stain or CAM stain and then heated toshow reducible sugars. The liquefied starch was partially hydrolyzed toglucose in cases where activity was observed.

Example 11: Starch Liquefaction Using Amylases and/or Glucoamylases ofthe Invention

This example describes an exemplary method of the invention forliquefying starch using amylases and/or glucoamylases of the invention.

Amylase concentrate can be prepared from fermentation broths by heattreatment, cell washing, alkaline extraction using microfiltration andultrafiltration (48% overall yield). The UF concentrate can beneutralized with acetic acid and formulated with 30% glycerol at pH 4.5.The activity level of the slurry formulation can be representative of acommercial product (120 U^(l)/g-0.5 kg/ton starch).

Standard Amylase Activity Assay

A 1 mL cuvette containing 950 μL of 50 mM MOPS pH 7.0 containing 5 mMPNP-α-D-hexa-(1→4)-glucopyranoside is placed in the Peltier temperaturecontroller of the Beckman DU-7400 spectrophotometer preheated to 80° C.The spectrophotometer is blanked at 405 nm and 50 μL of the enzymesolution is added to the cuvette, mixed well and the increase in theOD_(405 nm) is monitored over a one-minute interval. TheΔOD_(405 nm/min) rate is converted to a standard unit of μmole/minutefrom the OD_(405 nm) response of 50 μL of 1 μmole/mL PNP in 950 mL 50 mMMOPS at pH 7.0-80° C. One standard unit of thermostable alpha amylase(DTAA) is equal to the amount of enzyme that will catalyze the releaseof 1 μmole/mL/minute of pNP under the defined conditions of the assay.

Standard Glucoamylase Activity Assay

A 1 mL cuvette containing 950 μL of 50 mM MOPS pH 7.0 containing 5 mMpNP-α-D-glucopyranoside is placed in a Peltier temperature controller ofa DU-7400™ spectrophotometer (Beckman) preheated to 60° C. Thespectrophotometer is blanked at 405 nm and 50 μL of the enzyme solutionis added to the cuvette, mixed well and the increase in the OD_(405 nm)is monitored over a one-minute interval. The ΔOD_(405 nm)/min rate isconverted to a standard unit of μmole/minute from the OD_(405 nm)response of 50 μL of 1 μmole/mL pNP in 950 mL 50 mM MOPS at pH 7.0-60°C. One standard unit of glucoamylase (DGA) is equal to the amount ofenzyme that will catalyze the release of 1 μmole/mL/minute of pNP underthe defined conditions of the assay.

Dextrose Equivalent Determination

The neocuproine assay method can be used to measure the DE. Selectedsamples can be measured by a procedure described herein, and/or by a GPCanalyst using the GPC Fehlings procedure.

Neocuproine Assay

A 100 μl sample is added to 2.0 ml of neocuproine solution A (40 g/Lsodium carbonate, 16 g/L glycine, 0.45 g/L copper sulfate). To this isadded 2.0 ml of neocuproine solution B (1.2 g/L neocuproinehydrochloride-Sigma N-1626). The tubes are mixed and heated in a boilingwater bath for 12 minutes; cooled, diluted to 10 ml volume with DI waterand the OD read at 450 nm on the spectrophotometer. The glucoseequivalent in the sample is extrapolated from the response of a 0.2mg/ml glucose standard run simultaneously.

The starch sample is diluted ˜1 to 16 with DI water with the exactdilution recorded. Ten milliliters of the diluted sample is added to 20mls of DI water. Ten milliliters of Fehlings solution A and B are addedto the diluted starch. The sample is boiled for 3 minutes and cooled onice. Ten milliliters of 30% KI and 10 ml of 6N H₂SO₄ is added. Thesolution is titrated against 0.1N sodium thiosulfate. The titrant volumeis recorded and used to calculate the DE.

Residual Starch Determination

Post-saccharification samples can be checked for residual starch usingthe Staley iodine procedure. Twenty grams of sample is weighed into alarge weigh dish. 45 μL of Iodine solution is added to the weigh dishand the starch solution is mixed well. Dark blue indicates the presenceof starch, a light blue-green indicates slight starch, light greenindicates a trace of starch and yellow-red, absence of starch. Iodinesolution is prepared by dissolving 21.25 grams of iodine and 40.0 gramsof potassium iodide in one liter of water.

Oligosaccharide Profile

Liquefaction and saccharification carbohydrate profiles can be measuredby HPLC (e.g., an AMINEX HPX-87C™ column (Bio-Rad) in calcium form −80°C.) using refractive index detection.

Gel Permeation Chromatography

The molecular weight distribution can be determined by chromatography,e.g., on a PL AQUAGEL-OH Column™ with mass detection by refractive index(Waters Model 2410). A Model T60™ (Viscotek) detector can be used forcontinuous viscosity and light scattering measurements.

Capillary Electrophoresis

A Beckman Coulter P/ACE MDQ™ Glycoprotein System for separation of APTSderivatized oligosaccharides on a fused silica capillary can be used; inone aspect, detection by laser-induced fluorescence is used.

Primary Liquefaction

Line starch directly from the GPC process is pumped into a 60 liter feedtank where pH, DS (dry solids) and calcium level can be adjusted beforeliquefaction. The amylase is added to the slurry. The 32% DS slurry ispumped at 0.7 liter/minute by a positive displacement pump to the jet—apressurized mixing chamber where the starch slurry is instantaneouslyheated to greater than 100° C. by steam injection. The gelatinizedpartially liquefied starch is pumped through a network of piping (stillunder pressure) to give the desired dwell time (5 minutes) attemperature. The pressure is released into a flash tank and samples canbe taken. Samples were taken in duplicate.

Secondary Liquefaction

The liquefied starch is collected in one liter glass bottles and held ina water bath at 95 C for 90 minutes.

Saccharification

Liquefied starch is cooled to 60° C., the pH adjusted to 4.5 and thesamples treated with glucoamylase. Saccharification progress can bemonitored over time, e.g., by HPLC.

The liquefied syrups produced with each amylase are adjusted toapproximately pH 2.5 with 6N HCl immediately after the 90 minutesecondary liquefaction to inactivate any residual amylase. The syrupsare then adjusted to pH 4.5, placed in a 60° C. water bath andsaccharified with three levels of glucoamylase. The extent ofsaccharification is monitored by HPLC at 18-88 hour time points.

The liquefied syrups are saccharified with the standard dosage −0.04% ofa double-strength glucoamylase—and two lower dosages (50% and 25%) tomonitor any differences in the saccharification progress.

Saccharification progress data can be analyzed by % dextrose developmentvs time, e.g., with 0.04% glucoamylases; or, % dextrose development vstime, e.g., with 0.02% glucoamylases.

Post-Saccharification Sugar Profile

Molecular Weight Distribution

The molecular weight distribution of syrups liquefied to DE's of 12 and18 by amylases and/or glucoamylases of the invention, and in someaspects, using controls, e.g., commercial enzymes, e.g., Bacilluslicheniformis or commercial Bacillus stearothermophilus, can be measuredby gel permeation chromatography using detection by refractive index,light scattering and viscosity. Both the B. licheniformis and B.stearothermophilus amylases generate a bimodal distribution—the primarypeak centered at 2000, a secondary peak at 32,000 with a shoulderextending past the 160,000 range. The lower molecular weight peakrepresents approximately 60% of the total mass of the sample. In someembodiments, amylases and/or glucoamylases of the invention can exhibita single peak at 2000 with very little above 30,000.

HPLC

The DE 12 and 18 syrups produced by amylases and/or glucoamylases of theinvention (and the control commercial enzymes, e.g., amylases fromBacillus licheniformis and/or commercial Bacillus stearothermophilusamylases) can be analyzed by HPLC. Both techniques produce fingerprintscharacteristic of each class of amylase; the oligosaccharide patternsare different for B. licheniformis amylase vs B. stearothermophilusamylase, and amylases and/or glucoamylases of the invention are alsoexpected to produce fingerprints characteristic of their enzyme classes.The liquefied syrups of the invention (e.g., syrups made by methods ofthe invention and/or made by enzymes of the invention) exhibit evidenceof greater branching in the oligosaccharides. HPLC only resolve theoligosaccharides in the <DP15 range—larger fragments are not visible inthese techniques. Bacillus amylases are known to liquefy starch in amanner such that the amylopectin fraction is hydrolyzed less extensivelythan the amylose fraction. These >DP30 amylopectin fragments arecontained in the high molecular weight fraction centered at 32,000 andconsequently, little evidence of branching is seen in the HPLC analysesof the Bacillus liquefied syrups. In one aspect, <DP15 oligosaccharidesin liquefied syrups made using amylases and/or glucoamylases of theinvention contain fragments from both amylose and amylopectin.

Example 12: Starch Liquefaction at Acidic Conditions Using Amylasesand/or Glucoamylases of the Invention

The invention provides methods for liquefying starch using amylasesand/or glucoamylases of the invention, including amylases active underacidic conditions, e.g., between about pH 4.0 and 5.0, e.g., pH 4.5. Inone embodiment, the conversion of starch to glucose can be catalyzed bythe sequence action of two enzymes: an endoamylase of the invention,e.g., an amylase, such as an alpha-amylases, and an exoamylase, e.g., aglucoamylase of the invention, to liquefy the starch (e.g., thehydrolysis of high molecular weight glucose polymers to oligosaccharidesconsisting of 2 to 20 glycose units, typically a dextrose equivalent of10 to 12, by an amylase of the invention), followed by saccharificationwith an exoamylase, e.g., a glucoamylase (which can be a glucoamylase ofthe invention).

In one aspect, processing is in a corn wet milling plant producing astarch slurry having a pH or about 4.0 to 4.5. In one aspect, the pH israised, e.g., to 5.8 to 6.0 before liquefaction to accommodate an alphaamylase with a low pH activity and stability (which can be an alphaamylase of the invention). In one aspect, amylases and/or glucoamylasesof the invention can liquefy starch at pH 4.5 to dextrose equivalentsranging from 12 to 18; in one aspect, using alpha Amylases and/orglucoamylases of the invention at levels of about 3 to 6 grams per tonof starch. In this aspect, use of alpha Amylases and/or glucoamylases ofthe invention enable starch liquefaction to be conducted at pH 4.5.

In one aspect, starch liquefaction is conducted at pH 4.5 for 5 minutesat 105° C. to 90 minutes at 95° C. using amylases and/or glucoamylasesof the invention. The quantity of enzyme can be adjusted in order toadjust a target DE of 12 to 15 after liquefaction. In one aspect, theliquefied starch is then saccharified with a glucoamylase, e.g., anAspergillis glucoamylase, for about 48 hours at about pH 4.5 and 60° C.If the saccharified syrup does not contain at least 95% glucose, thetarget liquefaction DE is raised and the saccharification repeated untilthe liquefaction eventually does produce a saccharified syrup containingmore than 95% glucose. The amylase protein required to produce asuitable liquefied feedstock for saccharification can be determined,e.g., by PAGE or HPLC.

Example 13: Starch Liquefaction Using Amylases and/or Glucoamylases ofthe Invention

This example describes an exemplary method for liquefying starch usingamylases and/or glucoamylases of the invention; and described use ofcommercial Bacillus licheniformis and Bacillus stearothermophilusamylases as controls. These assays can compare the saccharificationprogress and final dextrose levels from syrups generated by enzymes ofthe invention and commercial amylases.

Dextrose equivalent (DE) is the industry standard for measuring theconcentration of total reducing sugars, calculated as D-glucose on a dryweight basis. Unhydrolyzed granular starch has a DE of virtually zero,whereas the DE of D-glucose is defined as 100. One exemplary process ofthe invention uses an enzyme dosage of about 60 to 70 Units/kilo starchat pH 4.5 to reach a 19 DE.

Oligosaccharide patterns generated by amylases and/or glucoamylases ofthe invention and commercial amylases can be analyzed by molecularweight (MW) distribution using, e.g., gel permeation chromatography withdetection by light scattering and viscosity. In one aspect, amylasesand/or glucoamylases of the invention can generate an 18DE and a uniformoligosaccharide MW distribution, with nothing greater than 20,000. Thisis consistent with the lower viscosity for syrups of the invention(e.g., syrups made by methods of the invention, or, made using enzymesof the invention). The DP (degrees of polymerization) profiles asmeasured by HPLC also can be used to analyze differences in actionpattern.

Amylase concentrate are prepared from fermentation broths by heattreatment, cell washing, alkaline extraction using microfiltration andultrafiltration (UF). The UF concentrate is neutralized with acetic acidand formulated with 30% glycerol at pH 4.5. The activity level of theslurry formulation can be analyzed, e.g., 120 Ul/g-0.5 kg/ton starch isrepresentative of a commercial product.

Example 14: Alkaline Amylases for Laundry and Autodishwash Applications

In one aspect, the invention provides detergents comprising amylasesand/or glucoamylases of the invention, including amylases and/orglucoamylases active under alkaline conditions, and methods of makingand using them.

The invention provides alkali-stable amylase and/or glucoamylaseenzymes, which can be compared to commercial benchmark enzyme(s) withrespect to features important in laundry and automatic dishwashing (ADW)applications:

-   -   An ADW wash test on starch-coated slides.    -   Amylase and/or glucoamylase enzyme activity testing in the        presence of a laundry/ADW formulation using a soluble substrate.    -   In the presence of chelators, amylase and/or glucoamylase enzyme        activity testing.    -   Amylase and/or glucoamylase enzyme activity testing and alkaline        pH optima ranges determined (e.g., from pH 10 to 11).    -   Amylase and/or glucoamylase enzyme activity testing for        thermophilic properties, e.g., performance at about 65° to 70°        C.

Amylase and/or glucoamylase activity can be measured either by areducing sugar assay or by monitoring the fluorescence at 520 nm (485 nmexcitation) when BODIPY-starch was used. Initial rates can be calculatedand converted to a percentage of the maximum rate.

Application Testing

Experiments can be designed to assess the activity and stability ofalkaline amylases and/or glucoamylases of the invention in laundry/ADWformulations and with the components individually. Amylase and/orglucoamylase activity can be assessed under conditions comprising thechelator EDTA and/or hydrogen peroxide; commercial benchmark enzymes canbe controls.

For example, purified proteins are incubated at 50° C. in the presenceor absence of 5 mM EDTA for a desired time(s), after which residualamylase activity is measured using soluble substrate. Activity in thepresence of EDTA is expressed as the % of activity in the absence ofchelator. Alternatively, enzyme activity in the presence of peroxidehydroxide can be assessed. Purified proteins are incubated at 50° C. inthe presence or absence of 1M H₂O₂ for a desired time, after whichamylase activity is measured using soluble starch. Activity in thepresence of peroxide hydroxide is presented as the % of activity in theabsence of H₂O₂. Enzyme activity can be tested in an ADW solution(distilled water, hardening solution, bleach, chelators, surfactants)with soluble substrate (BODIPY-starch). Purified proteins can be reactedwith soluble starch at 40° C. in the presence of laundry/ADWformulation. Initial rates are calculated over 5 minutes and expressedas fluorescent units (FU)/s per ng of protein.

Wash tests with starch-coated slides can be performed as follows:purified proteins are incubated with slides at 50° C. for 30 min in thepresence of ADW solution (distilled water, water hardening solution,bleach, chelators, surfactants). Starch removal is measured comparingweight loss after the enzyme treatment to the initial weight of theslide.

Characterization of Exemplary Amylases

The gene encoding the amylase can be modified to comprise aStarch/Carbohydrate Binding Domain. The proteins can be expressed withand without a C-terminal histidine tag, and in non-glycosylating and aglycosylating host. Enzymes can be expressed in Host/His tagcombinations, and pH and temperature optimas determined. Enzymesexpressed in a glycosylating host with a His tag can be used for testexperiments. The presence of the His tag should not affect specificactivity, however, glycosylation may result in a slightly lower specificactivity than that without glycosylation.

Example 15: Identification and Characterization of a ThermostableAmylase

The following example describes exemplary protocols for theidentification and characterization of thermostable amylases.

In one study, 350 fungal isolates were screened on solid mediumcontaining granular starch as the sole source of carbon. Strains thatcompletely hydrolyzed starch or displayed significant growth weresubmitted for cDNA isolation and proteomics analysis of their secretedproteins. A combination of Sequence Based Discovery and ProteomicAnalysis was employed for recovering DNA sequences encoding amylases,glucoamylases and glucosidases that were shown by proteomics to besecreted during growth on granular starch.

The recovered full-length cDNA sequences were subcloned for expressionin Pichia pastoris and the expressed proteins were further characterizedwith the BCA assay (which was used to determine the increase inconcentration of reducing ends during hydrolysis of starch by theamylases) and the Glucose Oxidase (GO) assay (which was used fordetecting glucose released from starch by the glucoamylases), exemplaryprotocols for both of these assays are described herein.

TABLE 1 Source (as Enzyme activity determined by 18S SEQ ID NOs: classCBM20 RNA) SEQ ID NO: 50 (encoded amylase no Aspergillus terreus by,e.g., SEQ ID NO: 49) SEQ ID NO: 52 (encoded amylase CBM20 Aspergillusterreus by, e.g., SEQ ID NO: 51) SEQ ID NO: 54 (encoded amylase noAspergillus terreus by, e.g., SEQ ID NO: 53) SEQ ID NO: 4 (encoded by,amylase CBM20 Cochliobolus e.g., SEQ ID NO: 3) heterostrophus SEQ ID NO:2 (encoded by, amylase CBM20 Cochliobolus e.g., SEQ ID NO: 1)heterostrophus SEQ ID NO: 32 (encoded amylase no Penicillium by, e.g.,SEQ ID NO: 31) chrysogenum 100% SEQ ID NO: 46 (encoded amylase noFusarium equiseti by, e.g., SEQ ID NO: 45) 100% SEQ ID NO: 22 (encodedamylase no Penicillium by, e.g., SEQ ID NO: 21) expansum 99% SEQ ID NO:24 (encoded amylase no Penicillium by, e.g., SEQ ID NO: 23) chrysogenum100% SEQ ID NO: 8 (encoded by, glucoamylase no Cochliobolus e.g., SEQ IDNO: 7) heterostrophus SEQ ID NO: 16 (encoded glucoamylase CBM20Cochliobolus by, e.g., SEQ ID NO: 15) heterostrophus SEQ ID NO: 14(encoded glucoamylase CBM20 Fusarium by, e.g., SEQ ID NO: 13)verticillioides GZ3639 SEQ ID NO: 18 (encoded glucoamylase CBM20Fusarium by, e.g., SEQ ID NO: 17) verticillioides GZ3639 SEQ ID NO: 10(encoded glucoamylase no Penicillium by, e.g., SEQ ID NO: 9) expansum99% SEQ ID NO: 12 (encoded glucoamylase CBM20 Fusarium equiseti by,e.g., SEQ ID NO: 11) 100% SEQ ID NO: 26 (encoded glucoamylase CBM20Penicillium by, e.g., SEQ ID NO: 25) verruculosum 100% SEQ ID NO: 20(encoded glucoamylase no Penicillium by, e.g., SEQ ID NO: 19)chrysogenum 100% SEQ ID NO: 28 (encoded glucoamylase CBM20 Fusarium by,e.g., SEQ ID NO: 27) merismoides 99% SEQ ID NO: 30 (encoded glucoamylaseCBM20 Phoma herbarum by, e.g., SEQ ID NO: 29) 99 SEQ ID NO: 34 (encodedglucoamylase no Penicillium herquei by, e.g., SEQ ID NO: 33) 99% SEQ IDNO: 36 (encoded glucoamylase CBM20 Fusarium by, e.g., SEQ ID NO: 35)oxysporum 100% SEQ ID NO: 38 (encoded glucoamylase CBM20 Cordyceps by,e.g., SEQ ID NO: 37) ophioglossoides 99% SEQ ID NO: 40 (encodedglucoamylase CBM20 Penicillium by, e.g., SEQ ID NO: 39) chrysogenum 100%SEQ ID NO: 42 (encoded glucoamylase no Cucurbitaria by, e.g., SEQ ID NO:41) berberidis 98% SEQ ID NO: 48 (encoded glucoamylase CBM20 Aspergillusby, e.g., SEQ ID NO: 47) versicolor 99 SEQ ID NO: 44 (encodedα-glucosidase Cochliobolus by, e.g., SEQ ID NO: 43) heterostrophus SEQID NO: 6 (encoded by, α-glucosidase Cochliobolus e.g., SEQ ID NO: 5)heterostrophus

Fungal isolates were screened on modified solid media according toMarlida (2000) World J. Microbiol. Biotechnol. 16:573-578. The mediumcontained Czapek Dox salts and 1% MIMAIZE²⁶⁰™ (HiMaize²⁶⁰, NationalStarch & Chemical, Bridgewater, N.J.) resistant starch as the solesource of carbon. The MIMAIZE²⁶⁰™ starch was added to the cool agarwithout sterilization in order to preserve the granular structure of thestarch. Additionally the same fungal strains were screened on a mediumwith 0.5% red starch (Megazyme, Ireland) in order to identify strainsthat secreted amylolytic enzymes.

After 5 days of growth, isolates that grew well on resistant starch andsecreted starch—degrading enzymes, visualized by a clearing zone on thered starch medium, were chosen as “primary hits”. In the second step,primary hits were grown in liquid medium with Czapek Dox salts and 1%resistant starch (MIMAIZE²⁶⁰™) as the sole source of carbon forapproximately 2 weeks. Strains that completely cleared the starchsolution or displayed significant growth in this medium were submittedfor cDNA isolation and proteomics analysis of culture supernatants. Thecombination of Sequence Based Discovery and proteomics was employed forrecovering sequences encoding amylases and glucoamylases.

Sequence Based Discovery was performed using two approaches. In one,universal degenerate primers for PCR recovery of amylase andglucoamylase genes were designed based on alignment of known fungalprotein sequences. In the other approach, universal degenerate primerswere combined in PCR with primers based on the peptide sequencesobtained from the proteomics analysis of culture supernatants fromfungal isolates grown on granular starch. In both cases the primers wereused for PCR with template DNA comprising cDNA made from the samestarch-digesting fungal strains. By this means, partial gene sequenceswere recovered. Peptide data were also used to facilitate recovery offull-length versions of the partial sequences using 5′ and 3′ RACE PCR.

For known fungal species with pre-sequenced genomes, proteomics analysiswas used to determine the sequences of peptides derived from putativeamylases using the following approach: 1) Following SDS-PAGE analysis ofsamples derived from fungal extracts grown on resistant starch as thesole source of carbon, individual bands were subjected to proteasedigestion; 2) Mass spectroscopy analysis of recovered peptides wascarried out on an LCQ instrument; 3) Peptide sequences were determinedby SEQUEST™ (Sage-N Research, Inc. and Thermo Scientific) databasesearches.

For unknown fungal species, the peptide sequences where determined by amethod similar to the one described above with the exception that theaccurate masses of peptides and their constituent amino acids weredetermined using a QTOF instrument. The sequences of the recoveredpeptides were used as a template to produce degenerate oligonucleotideprimers for PCR amplification of the genes from cDNA made from therelevant fungal species grown on MIMAIZE²⁶° ™ granular starch.

The recovered full-length cDNA sequences encoding amylases andglucoamylases were subcloned for expression in Pichia pastoris and theexpressed proteins were further characterized using the BCA assay formeasuring the increase in concentration of reducing ends during starchhydrolysis by amylases, and the GO assay for the detection of glucosereleased during starch hydrolysis by glucoamylases.

For determining the activity of both amylases and glucoamylases, starchhydrolysis reactions were performed as follows: Assays were performed intriplicate in an Eppendorf tabletop incubator with constant shaking (800rpm), at 37° C. and pH 5.0 in 50 mM sodium acetate buffer containing 1%raw granular starch. Reactions were started by adding the enzyme to thereaction mix. At different time points aliquots of the reactions werewithdrawn and quenched either by addition of 1M Tris pH 7.5(glucoamylases) or BCA reagent (amylases).

BCA Assay for Measuring the Increase in Concentration of Reducing Ends.

The activity of alpha-amylases was measured by the appearance ofreducing groups formed during the hydrolysis of starch. TheBCA—Bicincochinic Acid (Copper-BCA) assay of Reducing Sugars wasperformed according to Wong (2000) Microassay for rapid screening ofalpha-amylase activity, J. Agric. Food Chem. 48:4540-4543; and, Fox(1991) Miniaturization of three carbohydrate analyses using a microsample plate reader, Anal. Biochem. 195:93-96, was used.

A 10 μl aliquot of amylase starch hydrolysis reaction was quenched into100 μl of BCA reagent (consisting of 64 mg/mL sodium carbonatemonohydrate, 24 mg/mL sodium bicarbonate, 1.95 mg/mL BCA, 1.24 mg/mLcupric sulfate pentahydrate, 1.26 mg/mL L-serine). Color developmentoccurred during incubation of the quenched reaction at 80° C. for 35minutes, and was followed by absorbance determination at 560 nm. Initialrates were calculated over a 50 min reaction time. A standard curveusing maltose was constructed to correlate A_(560 nm) with theconcentration of generated reducing sugars (nmoles). Specific activitywas expressed as nmoles/min/μg enzyme.

Glucose Oxidase/Peroxidase (GO) Assay for the Quantification of GlucoseReleased During Starch Hydrolysis:

The activity of glucoamylases was measured by the modified GO (coupledglucose oxidase) assay according to Bergmeyer, In Determination withGlucose Oxidase and Peroxidase; Bergmeyer, H. U., Ed.; Methods ofEnzymatic Analysis, 2nd Ed.; 1974; pp 1205-1212).

GO reactions were started by adding 10 μl of the quenched starchhydrolysis reaction to 90 μl of PBS containing glucose oxidase (0.1U/ml), peroxidase (0.25 U/ml) and 0.05 mM Amplex Red, in black Nunc 96well plates. Plates were kept at room temperature in the dark for 30 minprior to reading on a fluorescence plate reader with Ex/Em 545/590 nm. Astandard curve constructed with glucose was used to assess the amount ofglucose produced in the hydrolysis reactions. Initial rates of starchhydrolysis (nmols of glucose released from 1% granular starch/min/μgglucoamylase) were determined by plotting the amount of glucose releasedover time, and calculating the slope of the best linear fit through thedata points.

The influence of pH in the range of between about pH 3.5 to 6.0 on thehydrolysis of starch by seven (7) exemplary glucoamylases and twoexemplary amylases of this invention is illustrated in FIG. 10 and FIG.11. FIG. 10 illustrates data showing the influence of pH on granularstarch hydrolysis by seven (7) exemplary glucoamylases of this inventionat 37° C.; initial rates were calculated over 15 min and converted tothe percentage of the maximum observed rate. FIG. 11 illustrates datashowing the influence of pH on granular starch hydrolysis by SEQ ID NO:4(encoded by, e.g., SEQ ID NO:3) and SEQ ID NO:52 (encoded by, e.g., SEQID NO:51) amylases at 37° C.; initial rates were calculated over 50 minand converted to the percentage of the maximum observed rate.

Other assays can also be used to characterize an enzyme of theinvention, and some exemplary protocols are described, below:

Exemplary Nucleic Acid Extraction Protocol: Microorganisms, e.g.,filamentous fungus, are grown in liquid culture. Biomass is collectedand high molecular weight genomic DNA is isolated using DNEASY™ (DNeasy)Plant Maxi Kit (Qiagen, Valencia, Calif.) using standard protocols.Total RNA can be isolated using RNEASY™ (RNeasy) Plant Mini Kit (Qiagen)using standard protocols.

Exemplary Library Construction Protocol: genomic DNA can be partiallydigested with restriction enzymes and fragments between 1-10 kb can bepurified for construction of a genome library. The fragments can beligated into the vector Lambda Zap Express™ (Stratagene, San Diego,Calif.) and packaged into infectable phage as per manufacturer'sinstructions.

Exemplary Library Screening Protocol: Lambda libraries can be used toinfect XL1 Blue MRF™ cells (Stratagene) in top agar. Approximately50,000 pfu of phage can be added to 600 ul of cells OD600=1. The mixtureis incubated at 37° C. for 15 minutes in a water bath and then added to6 ml melted 0.7% top agar and plated onto NZY agar plates. The plate isthen incubated overnight at 39° C. A nylon circle (F. Hoffmann-La RocheLtd., Basel Switzerland) can be laid on top of the resulting plaque lawnand lifted back up with some of the phage adhering to the nylon. Thenylon can be submerged in 1.5M NaCl, 0.5M NaOH for 2 minutes, 1.5M NaCl,0.5M Tris pH 7.6 for 5 minutes and 2×SSC, 0.2M Tris pH7.6 for 30seconds. The nylon filter is then UV crosslinked, e.g., in a Stratagenecrosslinker.

PCR fragments from amylase, e.g., glucoamylases, genes can be usedprobes, e.g., using an Expand High Fidelity PCR Kit™ (Roche) using 30cycles of 95° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 1minute in a thermal cycler. The isolated PCR fragment can be prepared asa radioactive probe using the Prime It Kit™ (Stratagene) followingmanufacturer's instructions.

The library filter lifts are washed in a prehybridization solution(e.g., DIG EASY HYB™, Roche) for two hours at 42° C. in a hybridizationoven (Robbins). The probe can be added to 15 ml fresh DIG EASY HYB™ andused to replace the prehybridization solution. The filter is washed withprobe overnight, e.g., at 45° C. The probe can be removed and the filterwashed once with 2×SSC, 0.1% SDS for 15 minutes, and twice with 0.1×SSC,0.1% SDS for 15 minutes each. The nylon filter can be exposed to x-rayfilm overnight at −80 C. Following developing, hybridization spots onthe x-ray film can be used to identify clones from the original plate.An agar plug can be taken from the plate where the spots lined up andsuspended in SM buffer to release the phage into solution. Severalisolated plaques corresponding to genomic fragments containing all orpart of an amylase gene can be thus isolated.

100 ul of isolated phage stock can be added to 200 ul XL-1 BLUE MRF™cells (Stratagene) and 1 ul EXASSIST™ helper phage (Stratagene). Themixture can be incubated at 37 C for 15 minutes, and 3 ml of 2×YT mediacan be added. This can be incubated at 37° C. with shaking for 2.5hours. The mix can be heated for 20 minutes at 70° C. and cooled on ice.100 ul of the mix can be removed and added to 200 ul SOLR cells(Stratagene) and incubated at 37° C. for 15 minutes. 50 ul can be platedon LB kanamycin (50 ug/ml) plates and incubated overnight at 37° C.Resulting colonies may contain cloned genomic fragments in the plasmidpBK-CMV.

Exemplary Sequencing Protocol: DNA sequencing on candidate clones can beperformed with the BIGDYE TERMINATOR™ cycle sequencing VERSION 2.0 Kit™(Applied Biosystems, Foster City, Calif.) and a 3700 DNA Analyzer™(Applied Biosystems) using manufacturer's protocols. Potential intronscan be identified by comparing this sequence with consensus sequencesfor introns in known amylases.

Exemplary cDNA Synthesis Protocol: PCT primers are used in a cDNAsynthesis reaction using a THERMOSCRIPT™ rtPCR Kit™ (Invitrogen) usingmanufacturer's protocols.

Exemplary Expression Cloning Protocol: PCT primers are used to generatea PCR fragment using the cDNA clone as a template using 30 cycles of 95°C. for 20 seconds, 55° C. for 30 seconds, 72° C. for 2 minutes, usingEXPAND HIGH FIDELITY PCR Kit™ (Roche) and manufacturer's protocols. ThePCR fragments are digested with the restriction enzymes and ligated intothe corresponding restriction sites of a plasmid, e.g., pPIC Z™ alpha(Invitrogen). The construct can be transformed into a yeast, e.g.,Pichia pastoris Strain X-33™ (Invitrogen) where the construct integratesstably into the Pichia chromosome. Selection can be based on resistanceto zeocin. The construct can be designed such that the Pichia clone canbe induced with methanol to secrete the mature amylase into the media. A1-liter culture of the expression clone can be inoculated with anovernight yeast starter culture in BMGY and grown overnight at 30° C. ina shake flask. The yeast cells are collected by centrifugation thefollowing day and resuspended in 1 liter of BMMY. The cells are culturedat 30° C. in a shake flask for 3 days with methanol added to 0.5% finalevery 24 hours. The media containing the expressed glucoamylase enzymeare then collected and tested in a glucoamylase activity assay and SDSPAGE electrophoresed using standard protocols to determine the proteinsize.

Primers also can designed for overexpression in Escherichia coli. PCRprimers are used to generate a PCR product as before, from the cDNAtemplate. The PCR fragment can be digested with the restriction enzymesand ligated into corresponding restriction sites of the plasmid, e.g., apSE420 (Invitrogen). The construct can be transformed into Escherichiacoli, e.g., Strain XL-1 Blue MR (Stratagene). Selection for the plasmidcan be based on ampicillin resistance. The amylase gene can be under thecontrol of a lac-z promoter and can be induced with IPTG(isopropyl-thio-galactopyranoside). The construct can be designed suchthat the mature glucoamylase gene will be expressed within theEscherichia cell and will contain an extra methionine residue at theN-terminus.

Exemplary “Standard” assay: Enzyme aliquots can be added to a solutionof 5 mM buffer, 3 mM malto-oligosaccharides (Sigma, M-3639) in awaterbath. 100 ul aliquots can be removed at time points to 200 ulglucose oxidase reagent (Sigma, GAGO-20) and incubated 37° C., 30 min.The reaction can be stopped with addition of 12 N sulfuric acid and theabsorbance at 540 nm determined. The full-length version of the enzymecan be tested for pH, temperature and substrate utilization.

Exemplary “Activity” Assay: Enzyme activity can be measured by therelease of free glucose from an oligo-dextrin substrate. The liberatedglucose can be oxidized in a coupled reaction resulting in a coloredproduct. An enzyme aliquot can be added to solution of 5 mM buffer, 3 mMmalto-oligosaccharides (Sigma, M-3639) in a water bath. 100 ul aliquotscan be removed at time points to 200 ul glucose oxidase reagent (Sigma,GAGO-20) and incubated 37° C., 30 min. The reaction is stopped withaddition of 12 N sulfuric acid and the absorbance at 540 nm determined.Time points are then plotted to determine the relative rate for thereaction. pH Profile: Acetate buffer (pH 4.0, 4.5, 5.0, and 5.4) as wellas phosphate buffer (pH 6.2, 7.0, 8.1) can be used in an activity assayto determine the relative rate for the glucoamylase at each pH.

Temperature Profile: The relative rate of the enzyme at varioustemperatures (e.g., 50° C., 60° C., 70° C., 80° C., and 85° C.) can bedetermined in acetate buffer pH 5.3.

Temperature Stability Data: Enzyme can be added to 5 mM acetate bufferat a desired indicated temperature. Enzyme aliquots can be removed toice at 4 minute intervals. The aliquots are then tested for activity onsubstrate for 20 minutes at 70° C.

Substrate Utilization: The dextrins maltose, maltotriose, panose,maltotetraose, and maltoheptaose can be substituted for themalto-oligosaccharides in the activity assay to test for substrateutilization of an amylase, e.g., a glucoamylase. Rate of glucose releasefor various substrates can be tested in 5 mM acetate buffer, 70° C.

Example 16: Amylase Activity Assay: BCA Reducing Ends Assay

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention, for example, by a BCAreducing ends assay. Amylase (including, e.g., glucoamylase) activitycan be determined using the following methodology.

1. Prepare 2 substrate solutions, as follows:

-   -   a) 2% soluble starch (potato or granular corn starch) pH 8        solution by dissolving 2 gm potato starch in 100 ml 100 mM        sodium phosphate pH 8).    -   b) 2% soluble starch (potato) pH 10 solution by dissolving 2 gm        potato starch in 100 ml 100 mM sodium carbonate.

Heat both solutions in a boiling water bath, while mixing, for 30-40minutes until starch dissolves.

2. Prepare Solution A from 64 mg/ml sodium carbonate monohydrate, 24mg/ml sodium bicarbonate and 1.95 mg/ml BCA(4,4′-dicarboxy-2,2′-biquinoline disodium salt (Sigma Chemical cat #D-8284). Added above to dH2O.

3. Prepare solution B by combining 1.24 mg/ml cupric sulfatepentahydrate and 1.26 mg/ml L-serine. Add mixture to dH₂O.

4. Prepare a working reagent of a 1:1 ration of solutions A and B.

5. Prepare a Maltose standard solution of 10 mM Maltose in dH2O, wherethe 10 mM maltose is combined in 2% soluble starch at desired pH to afinal concentration of 0, 100, 200, 300, 400, 600 μM. The standard curvewill be generated for each set of time-points. Since the curve isdetermined by adding 10 ul of the standards to the working reagent itworks out to 0, 1, 2, 3, 4, 6 nmole maltose.

6. Aliquot 1 ml of substrate solution into microcentrifuge tubes,equilibrate to desired temperature (5 min) in heat block or heated waterbath. Add 50 ul of enzyme solution to the inside of the tube lid.

7. While solution is equilibrating mix 5 ml of both solution A & B.Aliquot 100 ul to 96 well PCR plate. Set plate on ice.

8. After 5 minute temperature equilibration, close lid on tubes, invertand vortex 3 times. Immediately aliquot 10 ul into plate as t=0 (zerotime point). Leave enzyme mixture in heat block and aliquot 10 ul ateach desired time point (e.g. 0, 5, 10, 15, 20, 30 minutes).

9. Ensure that 12 wells are left empty (only working reagent aliquotted)for the addition of 10 ul of standards, for the standard curve.

10. When all time points are collected and standards are added, coverplate and heated to 80° C. for 35 min. Cool plate on ice for 10 min. Add100 ul H₂O to all wells. Mix and aliquot 100 ul into flat bottomed96-well plate and read absorbance at 560 nm.

11. Zero each sample's time points against its own t=0 (subtract theaverage t=0 A560 value from other average A560 values). Convert theA560_((experimental)) to umole (Divide A560_((experimental)) by theslope of the standard curve (A560/umole). Generate a slope of the timepoints and the umole (in umole/min), multiply by 100 (as the umole valueonly accounts for the 10 ul used in the assay, not the amount made inthe 1 ml rxn). To get the specific activity divide the slope (inumole/min) by the mg of protein. All points should be done at a minimumin duplicate with three being best. Divide protein concentration (mg/ml)by any dilution to get mg used in assay. Divide the above slope by mgused in assay to get specific activity. See for example, Wong (2000) J.Agric. Food Chem. 48:4540-4543; Fox (1991) Anal. Biochem. 195, 93-96.

Example 17: Screening for Amylase Activity

The following example describes an exemplary method for determining if apolypeptide is within the scope of the invention. Amylase (e.g.,glucoamylase) activity of clones can be assessed by a number of methodsknown in the art. The following is an example of methodology that can beused.

The number of plaques screened, per plate, can be approximately 10,000pfu's. For each DNA library: about 50,000 plaques per isolated libraryand 200,000 plaques per non-isolated library can be screened dependingupon the pfu titer for the λ Zap Express amplified lysate.

Titer Determination of Lambda Library

-   -   1) μL of Lambda Zap Express amplified library stock added to 600        μL E. coli MRF′ cells (OD₆₀₀=1.0). To dilute MRF′ stock, 10 mM        MgSO₄ is used.    -   2) Incubate at 37° C. for 15 minutes.    -   3) Transfer suspension to 5-6 mL of NZY top agar at 50° C. and        gently mix. Immediately pour agar solution onto large (150 mm)        NZY media plate.    -   4) Allow top agar to solidify completely (approximately 30        minutes), then invert plate.    -   5) Incubate the plate at 39° C. for 8-12 hours.    -   6) Number of plaques is approximated. Phage titer determined to        give 10,000 pfu/plate.    -   7) Dilute an aliquot of Library phage with SM buffer if needed.

Substrate Screening

-   -   Lambda Zap Express (50,000 pfu) from amplified library added to        600 μL of E. coli MRF′ cells (OD600=1.0). For non-environment        libraries, prepare 4 tubes (50,000 pfu per tube).    -   Incubate at 37° C. for 15 minutes.    -   While phage/cell suspension are incubating, 1.0 mL of red starch        substrate (1.2% w/v) is added to 6.0 mL NZY top agar at 50° C.        and mixed thoroughly. Keep solution at 50° C. until needed.    -   Transfer ⅕ (10,000 pfu) of the cell suspension to substrate/top        agar solution and gently mixed.    -   Solution is immediately poured onto large (150 mm) NZY media        plate.    -   Allow top agar to solidify completely (approximately 30        minutes), then invert plate.    -   Repeat procedures 4-6 four times for the rest of the cell        suspension (⅕ of the suspension each time).    -   Incubate plates at 39° C. for 8-12 hours.    -   Plate observed for clearing zones (halos) around plaques.    -   Plaques with halos are cored out of agar and transferred to a        sterile micro tube. A large bore 200 μL pipette tip works well        to remove (core) the agar plug containing the desired plaque.    -   Phages are re-suspended in 500 μL SM buffer. 20 μL chloroform is        added to inhibit any further cell growth.    -   Pure phage suspension is incubated at room temperature for 4        hours or overnight before next step.

Isolation of Pure Clones

-   -   10 μL of re-suspended phage suspension is added to 500 μL of E.        coli MRF′ cells (OD600=1.0).    -   Incubate at 37° C. for 15 minutes.    -   While phage/cell suspension is incubating, 1 mL of red starch        substrate (1.2% w/v) is added to 6.0 mL NZY top agar at 50° C.        and mixed thoroughly. Keep solution at 50° C. until needed.    -   Cell suspension is transferred to substrate/top agar solution        and gently mixed.    -   Solution is immediately poured onto large (150 mm) NZY media        plate.    -   Allow top agar to solidify completely (approximately 30        minutes), then invert plate.    -   Plate incubated at 39° C. for 8-12 hours.    -   Plate observed for a clearing zone (halo) around a single plaque        (pure clone). If a single plaque cannot be isolated, adjust        titer and re-plate phage suspension.    -   Single plaque with halo is cored out of agar and transferred to        a sterile micro tube. A large bore 200 μL pipette tip works well        to remove (core) the agar plug containing the desired plaque. To        amplify the titer, core 5 single active plaques into a micro        tube.    -   Phages are re-suspended in 500 μL SM buffer. 20 μL Chloroform is        added to inhibit any further cell growth.    -   Pure phage suspension is incubated at room temperature for 4        hours or overnight before next step. The pure phage suspension        is stored at −80° C. by adding DMSO into the phage suspension        (7% v/v).

Excision of Pure Clone

-   -   100 μL of pure phage suspension is added to 200 μL E. coli MRF′        cells (OD600=1.0). To this, 1.0 μL of EXASSIST™ helper phage        (>1×106 pfu/mL; Stratagene) is added. Use 2059 Falcon tube for        excision.    -   Suspension is incubated at 37° C. for 15 minutes.    -   3.0 mL of 2×YT media is added to cell suspension.    -   Incubate at 30° C. for at least 6 hours or overnight while        shaking.    -   Tube transferred to 70° C. for 20 minutes. The phagemid        suspension can be stored at 4° C. for 1 to 2 months.    -   100 μL of phagemid suspension transferred to a micro tube        containing 200 μL of E. coli Exp 505 cells (OD600=1.0).    -   Suspension incubated at 37° C. for 15 minutes.    -   300 μL of SOB is added to the suspension.    -   Suspension is incubated at 37° C. for 30 to 45 minutes.    -   100 μL of suspension is transferred to a small (90 mm) LB media        plate containing Kanamycin (LB media with Kanamycin 50 μg/mL)        for Zap Express DNA libraries or Ampicillin (LB media with        Kanamycin 100 μg/mL) for Zap II DNA libraries.    -   The rest of suspension is transferred to another small LB media        plate.    -   Use sterile glass beads to evenly distribute suspension on the        plate.    -   Plates are incubated at 30° C. for 12 to 24 hours.    -   Plate observed for colonies.    -   Inoculate single colony into LB liquid media containing suitable        antibiotic and incubate at 30° C. for 12 to 24 hours.    -   Glycerol stock can be prepared by adding 80% glycerol into        liquid culture (15% v/v) and stored at −80° C.

Activity Verification

-   -   50 μL of liquid culture is transferred to a micro tube. Add 500        μL of 8% pH 7 Amylopectin Azure into the same tube. Prepare 2        tubes for each clone.    -   Activity is tested at 50° C. for 3 hours and overnight. Use pH 7        buffer as control.    -   Cool the test specimen at ice-water bath for 5 minutes.    -   Add 750 μL of Ethanol and mixed thoroughly.    -   Centrifuge at 13000 rpm (16000 g's) for 5 minutes.    -   Measure OD of the supernatant at 595 nm.

RFLP Analysis

-   -   1.0 mL of liquid culture is transferred to a sterile micro tube.    -   Centrifuge at 13200 rpm (16000 g's) for 1 minute.    -   Discard the supernatant. Add another 1.0 mL of liquid culture        into the same sterile micro tube.    -   Centrifuge at 13200 rpm (16000 g's) for 1 minute.    -   Discard the supernatant.    -   Follow QIAPREP™ spin mini kit protocol for plasmid isolation.    -   Check DNA concentration using BioPhotometer.    -   Use Sac I and Kpn I for first double digestion. Incubate at        37° C. for 1 hour.    -   Use Pst I and Xho I for second double digestion. Incubate at        37° C. for 1 hour.    -   Add Loading dye into the digested sample.    -   Run the digested sample on a 1.0% agarose gel for 1-1.5 hours at        120 volts.    -   View gel with gel imager. All clones with a different digest        pattern will be sent for sequence analysis.

Example 18: Exemplary Protocols for Purifying Enzymes

The following example describes exemplary protocols for purifyingenzymes of this invention.

SEQ ID NO:52: 6 g of lyophilized supernatant of P. pastoris culture (seediscussion in Example 25, below) expressing SEQ ID NO:52 was suspendedin 24 mL of H₂O and precipitated with cold ethanol. Precipitated pelletwas re-suspended in approximately 40 mL of H₂O and dialyzed O/N againstwater. After dialysis a concentrated acetate buffer pH 6.0 was added tothe sample to get a final concentration of 50 mM. The protein bound tothe column (Q SEPHAROSE™; Amersham Pharmacia resin poured in a XK 50™column) in 50 mM Na acetate pH6.0 and was eluted during a gradientbetween 0 and 400 mM NaCl in Na acetate pH6.0. Contaminating proteinswere removed from the column with 1M NaCl in Na acetate pH6.0. Elutionof amylase from the Q SEPHAROSE™ was tracked with SDS PAGE and activityassays using BODIPY-starch as a substrate. Multiple purifications wererun to obtain enough protein to meet the requirement of approximately 1g. The purified fractions from these runs were pooled together andconcentrated by stir cell concentrator.

SEQ ID NO:48:

24 g of lyophilized supernatant of P. pastoris culture expressing SEQ IDNO:48 was suspended in 20 mL of 50 mM carbonate buffer pH 10.0; 100 mMNaCl and precipitated with cold ethanol. Precipitated pellet wasre-suspended in approximately 20 mL of 50 mM acetate buffer pH 5.2; 500mM NaCl, and dialyzed O/N against 50 mM malic buffer pH 3.5; 500 mMNaCl. The protein bound to the 100 mL agarose-amylose (NEB) column in 50mM malic buffer pH3.5; 500 mM NaCl and was eluted with 0.5% corn dextrinin 50 mM carbonate buffer pH 10.0; 50 mM NaCl. Contaminating proteinswere removed from the column with 50 mM sodium phosphate buffer pH 7.5;100 mM NaCl. Elution of glucoamylase from the agarose-amylose wastracked with SDS PAGE.

Other enzymes of the invention, including the exemplary enzymes, can bepurified using these protocols or variations thereof, or analogousprotocols.

FIG. 26 illustrates tables summarizing the efficiency of thesepurification protocols for the exemplary SEQ ID NO:4, SEQ ID NO:18, SEQID NO:26, and corresponding activity data on, inter alia, raw starch andsoluble starch comparing purified and unpurified enzyme.

Purified enzyme preparations of the exemplary SEQ ID NO:52 and SEQ IDNO:26 were analyzed for activity (the ability to hydrolyze) in rawstarch fermentations; this data on the purity (determined bydensitometric analysis), concentration, quantity and storage buffer issummarized:

SEQ ID NO: 52 SEQ ID NO: 48 Class Alpha-amylase Glucoamylase StorageBuffer GMP* GMA** Concentration  25 mg/ml  25 mg/ml Volume Sent  40 ml20 Total Amount 1.0 g 0.5 g Purity >90% >95% *GMP - 20% glucose; 0.1%methyl paraben, PBS pH 7.0 **GMA - 20% glucose; 0.1% methyl paraben; 50mM sodium acetate pH 5.2; 100 mM NaCl.

Example 19: Exemplary Protocol for Liquefying Starch and MeasuringResults

The following example describes exemplary protocols for liquefyingstarch using, e.g., enzymes of this invention. Reaction Conditions: 100mM PO₄ pH 6.5, 1% (w/w) liquefied starch DE 12 at 55° C. Both TLC andHPLC assays can be done to verify activity.

An exemplary protocol for the saccharification of liquefied starch at pH6.5:

-   -   Adjust the pH of the liquefied starch to the pH at which the        saccharification(s) will be performed. Liquefy starch in 100 mM        sodium acetate buffer, pH 4.5 with 100 mM sodium phosphate salts        added so that before saccharification, the pH could be adjusted        to pH 6.5.    -   Weigh 5 gram samples of liquefied starch into tared bottles.        -   Use 0.04% (w/w) OPTIDEX L-400™ or approximately 400 mL of            1-10 diluted stock OPTIDEX L-400™ per 100 grams of liquefied            starch.        -   Calculate the milligrams of OPTIDEX L-400™ contained in the            400 mL of 1-10 diluted stock OPTIDEX L-400™. Next, calculate            the volume of lysates needed to give the same concentration            of enzyme as the OPTIDEX L-400™.        -   Add enzymes to liquefied starch samples and incubate at            desired temperature)(50 C°. After 18 hours determine DE and            prepare a sample for HPLC analysis.

An Exemplary DE Determination:

Exemplary Neocuproine Assay:

A 100 ml sample can be added to 2.0 ml of neocuproine solution A (40 g/Lsodium carbonate, 16 g/L glycine, 0.45 g/L copper sulfate). To this canbe added 2.0 ml of neocuproine solution B (1.2 g/L neocuproinehydrochloride-Sigma N-1626). The tubes can be mixed and heated in aboiling water bath for 12 minutes; cooled, diluted to 10 ml volume withDI water and the OD read at 450 nm on the spectrophotometer. The glucoseequivalent in the sample can be extrapolated from the response of a 0.2mg/ml glucose standard run simultaneously.

Exemplary HPLC Analysis:

Saccharification carbohydrate profiles are measured by HPLC (Bio-RadAminex HPX-87A column in silver form, 80° C.) using refractive indexdetection. Mobile phase is filtered Millipore water used at a flow rateof 0.7 ml/min. Saccharification samples are diluted 1-10 with acidifiedDI water (5 drops of 6 M HCl into 200 mL DI water) then filtered througha 0.45 mm syringe filter. Injection volume is 20 uL.

Exemplary TLC:

Reaction products can be w/d at hourly timepoints and spotted and driedon a TLC plate. The plate can be then developed in 10:90water:isopropanol and visualized with either a vanillin stain or CAMstain and then heated to show reducible sugars. The liquefied starch canbe partially hydrolyzed to glucose in cases where activity was observed.

Example 20: Starch Liquefaction Using Glucoamylases

This example describes an exemplary method of the invention forliquefying starch using amylases and/or glucoamylases of the invention.Glucoamylase concentrate can be prepared from fermentation broths byheat treatment, cell washing, alkaline extraction using microfiltrationand ultrafiltration (48% overall yield). The UF concentrate can beneutralized with acetic acid and formulated with 30% glycerol at pH 4.5.The activity level of a commercial product can be about 120 U^(l)/g-0.5kg/ton starch.

Exemplary Glucoamylase Activity Assay

A 1 mL cuvette containing 950 μL of 50 mM MOPS pH 7.0 containing 5 mMPNP-α-D-hexa-(1→4)-glucopyranoside is placed in the Peltier temperaturecontroller of the Beckman DU-7400 spectrophotometer preheated to 80° C.The spectrophotometer is blanked at 405 nm and 50 μL of the enzymesolution is added to the cuvette, mixed well and the increase in theOD_(405 nm) is monitored over a one-minute interval. TheΔOD_(405 nm/min) rate is converted to a standard unit of μmole/minutefrom the OD_(405 nm) response of 50 μL of 1 μmole/mL PNP in 950 mL 50 mMMOPS at pH 7.0-80° C. One standard unit of thermostable alphaglucoamylase (DTAA) is equal to the amount of enzyme that will catalyzethe release of 1 μmole/mL/minute of pNP under the defined conditions ofthe assay.

Standard Glucoamylase Activity Assay

A 1 mL cuvette containing 950 μL of 50 mM MOPS pH 7.0 containing 5 mMpNP-α-D-glucopyranoside is placed in the Peltier temperature controllerof the Beckman DU-7400 spectrophotometer preheated to 60° C. Thespectrophotometer is blanked at 405 nm and 50 μL of the enzyme solutionis added to the cuvette, mixed well and the increase in the OD_(405 nm)is monitored over a one-minute interval. The ΔOD_(405 nm)/min rate isconverted to a standard unit of μmole/minute from the OD_(405 nm)response of 50 μL of 1 μmole/mL pNP in 950 mL 50 mM MOPS at pH 7.0-60°C. One standard Diversa unit of glucoamylase (DGA) is equal to theamount of enzyme that will catalyze the release of 1 μmole/mL/minute ofpNP under the defined conditions of the assay.

Dextrose Equivalent Determination

The neocuproine method is used to measure the DE. Selected samples weremeasured by both the procedure described above, and by a GPC analystusing the GPC Fehlings procedure.

Neocuproine Assay

A 100 μl sample is added to 2.0 ml of neocuproine solution A (40 g/Lsodium carbonate, 16 g/L glycine, 0.45 g/L copper sulfate). To this isadded 2.0 ml of neocuproine solution B (1.2 g/L neocuproinehydrochloride-Sigma N-1626). The tubes were mixed and heated in aboiling water bath for 12 minutes; cooled, diluted to 10 ml volume withDI water and the OD read at 450 nm on the spectrophotometer. The glucoseequivalent in the sample is extrapolated from the response of a 0.2mg/ml glucose standard run simultaneously.

The starch sample is diluted ˜1 to 16 with DI water with the exactdilution recorded. Ten milliliters of the diluted sample is added to 20mls of DI water. Ten milliliters of Fehlings solution A and B were addedto the diluted starch. The sample is boiled for 3 minutes and cooled onice. Ten milliliters of 30% KI and 10 ml of 6N H₂SO₄ is added. Thesolution is titrated against 0.1N sodium thiosulfate. The titrant volumeis recorded and used to calculate the DE.

Residual Starch Determination

Post-saccharification samples were checked for residual starch using theStaley iodine procedure.

Twenty grams of sample is weighed into a large weigh dish. 45 μL ofIodine solution is added to the weigh dish and the starch solution ismixed well. Dark blue indicates the presence of starch, a lightblue-green indicates slight starch, light green indicates a trace ofstarch and yellow-red, absence of starch. Iodine solution is prepared bydissolving 21.25 grams of iodine and 40.0 grams of potassium iodide inone liter of water.

Oligosaccharide Profile

Liquefaction and saccharification carbohydrate profiles were measured byHPLC (Bio-Rad AMINEX HPX-87C™ column in calcium form −80° C.) usingrefractive index detection.

Gel Permeation Chromatography

The molecular weight distribution is determined by chromatography on aPL AQUAGEL-OH™ column with mass detection by refractive index (WatersModel 2410). A Viscotek Model T60™ detector is used for continuousviscosity and light scattering measurements.

Capillary Electrophoresis

Beckman Coulter P/ACE MDQ™ Glycoprotein System—separation of APTSderivatized oligosaccharides on a fused silica capillary—detection bylaser-induced fluorescence.

Primary Liquefaction

Line starch directly from the GPC process is pumped into a 60 liter feedtank where pH, DS (dry solids) and calcium level can be adjusted beforeliquefaction. The glucoamylase is added to the slurry. The 32% DS slurryis pumped at 0.7 liter/minute by a positive displacement pump to thejet—a pressurized mixing chamber where the starch slurry isinstantaneously heated to greater than 100° C. by steam injection. Thegelatinized partially liquefied starch is pumped through a network ofpiping (still under pressure) to give the desired dwell time (5 minutes)at temperature. The pressure is released into a flash tank and samplescan be taken. Samples were taken in duplicate.

Secondary Liquefaction

The liquefied starch is collected in one liter glass bottles and held ina water bath at 95° C. for 90 minutes.

Saccharification

Liquefied starch is cooled to 60° C., the pH adjusted to 4.5 and thesamples treated with glucoamylase. Saccharification progress ismonitored over time by HPLC.

Saccharification

The liquefied syrups produced with each glucoamylase were adjusted toapproximately pH 2.5 with 6N HCl immediately after the 90 minutesecondary liquefaction to inactivate any residual glucoamylase. Thesyrups were then adjusted to pH 4.5, placed in a 60° C. water bath andsaccharified with three levels of glucoamylase. The extent ofsaccharification is monitored by HPLC at 18 to 88 hour time points.

The liquefied syrups were saccharified with the standard dosage—0.04% ofa double-strength glucoamylase—and two lower dosages (50% and 25%) tomonitor any differences in the saccharification progress.

Saccharification Progress—% dextrose development vs time—0.04%glucoamylase.

Example 21: Exemplary Starch Liquefaction Process

This example describes an exemplary starch liquefaction process of theinvention comprising use of enzymes of the invention. The conversion ofstarch to glucose can be catalyzed by the sequence action of twoenzymes: amylases (e.g., alpha-amylases), including enzymes of theinvention, to liquefy the starch (e.g., the hydrolysis of high molecularweight glucose polymers to oligosaccharides consisting of 2 to 20glycose units, typically a dextrose equivalent of 10 to 12, by aglucoamylase of the invention), followed by saccharification with aglucoamylase (which can be a glucoamylase of the invention). In oneaspect, processing is in a corn wet milling plant producing a starchslurry having a pH or about 4.0 to 4.5. In one aspect, the pH is raised,e.g., to 5.8 to 6.0 before liquefaction to accommodate a glucoamylasewith a low pH activity and stability. In one aspect, amylases and/orglucoamylases of the invention can liquefy starch at pH 4.5 to dextroseequivalents ranging from 12 to 18; in one aspect, using glucoamylases ofthe invention at levels of about 3 to 6 grams per ton of starch. In thisaspect, use of glucoamylases of the invention enables starchliquefaction to be conducted at pH 4.5.

In one aspect, starch liquefaction is conducted at pH 4.5 for 5 minutesat 105° C. to 90 minutes at 95° C. using glucoamylases of the invention.The quantity of enzyme is adjusted in order to adjust a target DE of 12to 15 after liquefaction. In one aspect, the liquefied starch is thensaccharified with a glucoamylase, e.g., an Aspergillis glucoamylase, forabout 48 hours at about pH 4.5 and 60° C. If the saccharified syrup didnot contain at least 95% glucose, the target liquefaction DE is raisedand the saccharification repeated until the liquefaction eventually didproduce a saccharified syrup containing more than 95% glucose. Theglucoamylase protein required to produce a suitable liquefied feedstockfor saccharification is determined by PAGE.

Example 22: Identification of Peptides Resulting from ProteaseProteolysis in Simulated Gastric Fluid (SGF) Test

This example describes the identification of small peptides resultingfrom pepsin proteolysis of the exemplary enzyme of the invention SEQ IDNO:52 (encoded, e.g., by SEQ ID NO:51). This example also describes theevaluation of activity of exemplary enzymes of the invention in in vitro“Simulated Gastric Fluid” (SGF) tests. The SGF tests showed that all theenzymes were quickly digested by the gastric protease pepsin. In onecase a small pepsin-resistant fragment was observed after 60 minutes oftreatment.

FIG. 21 illustrates an SDS PAGE showing the results of proteolysis (invitro digestibility, the SGF test) of SEQ ID NO:52 by pepsin at pH 1.3;pepsin cuts at the C-terminus of Lys (K) and Phe (F) residues (the upperarrow indicates a 36.5 K band, the middle arrow a 5 K band (seediscussion, below), and the lower arrow a 3 K band). SEEBLUE PLUS2™prestained Standard from Invitrogen, and MARKER12™ from Invitrogen wasused. All the samples were run on 16% Tricine gels. A common bandpresent in all tests is pepsin.

FIG. 22 illustrates the characterization of the peptides generated inthis digest of SEQ ID NO:52, as identified using LC MS/MS (LiquidChromatography/Mass Spectrometry/Mass Spectrometry) analysis; anN-linked glycosylation site is identified.

FIG. 23A illustrates the small peptide isolation scheme used (for thepeptides generated by the proteolysis of SEQ ID NO:52 by pepsin). FIG.23B is discussed, below, and illustrates an SDS PAGE of the results ofthe small peptide isolation scheme, where lane 1 is pepsin only, lane 2is undigested amylase SEQ ID NO:52, lane 3 is the amylase SEQ ID NO:52digested by pepsin, lane 4 is the 30 kd cut-off top as illustrated inFIG. 23A, lane 5 is the 30 kd flow-through as illustrated in FIG. 23A,lane 6 is the sample captured by C18 RP (C18 reverse phase columnchromatography) as illustrated in FIG. 23A.

FIG. 23C illustrates the LC/MS profile of the C18 RP eluted fraction,and is discussed, below.

FIG. 23D illustrates the sequence of the peptides identified by the LCMS/MS analysis; FIG. 23E and FIG. 23F illustrate the “Asn-Xaa-Ser/Thr”sequins (motifs) in the sequence output (highlighted in blue);asparagines predicted to be N-glycosylated are highlighted in red.

In Vitro Digestibility Assays:

No full-length versions of any of the enzymes tested were detected bySDS-PAGE after SGF treatment, indicating that pepsin had digested eachof the full-length proteins; after 60 minutes of SGF treatment, smallproteolytic fragments, about 6 kDa in size, were observed in digests ofalpha-amylase SEQ ID NO:52 (see discussion in Example 22).

Activity Test after Pepsin Treatment:

To determine if there is any residual activity remaining after pepsintreatment, the SGF test was modified and activity tests were performedwith dextrin as substrate for glucoamylases and BODIPY-starch assubstrate for alpha-amylases. No residual activity was observed for anyof the enzymes tested, after SGF treatment for 60 minutes. The loss ofactivity was caused either by pepsin digestion of the test proteins(e.g. SEQ ID NO:4, SEQ ID NO:76 and SEQ ID NO:2), by the inactivation ofthe enzyme under the low pH reaction conditions, or both.

To identify the pepsin resistant peptide band, migrating at 5 kDa rangeon a SDS-PAGE, small peptides where enriched by filtering the sampleover a 30 kDa filter membrane. The flow through was further purifiedusing a C18 solid phase extraction column. The recovered peptides wherethen analyzed by Edmond degradation N-terminal sequencing, LC-MS, and LCtandem mass spectroscopy to determine the peptide sequence. The samplesanalyzed by LC tandem mass spectroscopy were first treated with trypsinprotease.

Peptide Purification:

To determine the sequence of the 5 kDa peptide band, the peptide wasenriched by first passing the pepsin treated SEQ ID NO:52 reaction overa 30 KDa filter membrane. The flow-through was then purified using a C18solid phase extraction column. The recovered peptides formed a band atthe 5 kDa range on an SDS-PAGE. FIG. 23B illustrates an SDS-PAGEanalysis of the pepsin resistant alpha-amylase SEQ ID NO:52 peptide.This gel was Coomassie stained. Lane 1 is the pepsin protease. Lane 2 isthe amylase SEQ ID NO:52. Lane 3 is the amylase SEQ ID NO:52 treatedwith pepsin protease. Lane 4 is same as sample 3 which did not flowthrough a 30 kDa filter membrane. Lane 5 is the flow through of samplein lane 3 after passed through a 30 kDa filter membrane. Lane 6 issample 5 after recovery from a C18 solid phase extraction column.

To determine the peptide masses in the recovered sample the peptideswhere analyzed by LC-MS analysis over a C18 reverse phase column. Them/z profile of this sample showed that there are multiple masses ofvarious m/z values that are present in this sample, as illustrated inFIG. 23C, an LC-MS analysis of the amylase SEQ ID NO:52 small peptides.The solid phase was a C18 RP material. A gradient of 5%-80% ACN (15-90minutes) was used as the mobile phase in the experiment.

Edmond Degradation N-Terminal Sequencing:

To determine the sequence of the SEQ ID NO:52 small peptides, thepurified peptides where sent for N-terminal sequencing using Edmonddegradation method. The results for this analysis were inconclusive,since, a number of different amino acids were released at each cleavagestep. This result was consistent with the LC/MS analysis that showedthat there are more than one prominent species of peptides present inthis sample.

LC Tandem Mass Spectroscopy Analysis:

To determine the sequence of peptides present in the amylase SEQ IDNO:52 purified peptide fraction, these where treated with trypsinprotease and analyzed by LC MS/MS. This analysis resulted in theidentification of the following seven peptides; these peptide sequenceswere identified by SEQUEST™ searches from the LC MS/MS analysis of theamylase SEQ ID NO:52 small tryptic peptides:

Residue location Peptide Peptide sequence within SEQ ID NO: 52 1AGQEQHYSGGSDPANR 349-364 2 VFSGDPAYTCPYQN 251-264 3 SGDPAYTCPYQN 253-2644 SLLLLLSVFGQATHA   6-20 5 YENTGDGTSYHG  90-101 6 VYCGGSWQGIINHLD  56-707 GYSAGATLVETYTCT 448-462

It also determined that there were a number of peptide peaks in theLC-MS spectrum that their MS/MS profile did not match neither SEQ IDNO:52 nor other peptides in the database. One possible reason for thiscould be the complex N-linked glycosylation modification of the parentprotein that takes place in the host organism, Pichia pastoris. To testthis hypothesis, the extracted peptides were first treated with PNGase Fto remove their N-linked glycosyl groups. The resulting peptides wherethen trypsin treated and where subjected to LC MS/MS analysis todetermine their peptide composition. This analysis determined that therewhere at least 6 different peptide species present in the sample (seetable summary above). The resolution of these peptides, postdeglycosylation treatment, suggests that these peptides where part ofother peptides that where glycosylated in the sample.

In conclusion, N-terminal sequencing and LC-MS analysis showed that the5 KDa peptide band resulting from the pepsin protease digestion ofamylase SEQ ID NO:52 is composed of many different peptide species. Thisis most likely due to incomplete digestion of the amylase SEQ ID NO:52by the pepsin protease partly due to glycosylation state of thesepeptides. Thus, there is no one prominent peptide species in the MSspectra—multiple peptides are present; multiple different peptidesspecies were sequenced in the sample; N-terminal chemical sequencingalso provided evidence for the presence of multiple peptides; there isone peptide species that only appears after PNGase F deglycosylation ofthe sample, suggesting a glycosylation-related event is responsible forthe appearance of this peptide.

This exemplary series of protocols can be used on any polypeptide of theinvention, e.g., an exemplary sequence of the invention, to determinesequence, motifs, including glycosylation motifs, active sites and thelike.

Example 23: Low Temperature-Active Amylases

This example describes making and characterizing exemplary enzymes ofthe invention SEQ ID NO:56, SEQ ID NO:52; SEQ ID NO:62; SEQ ID NO:70,that are active at low temperatures, including having the ability tohydrolyze starch at low temperatures. This example also describes thedevelopment of an enzyme cocktail of the invention that canhydrolyze>95% of the starch in milled corn into fermentable sugars in nomore than 60 hours at about 30 to 40° C. and about pH 3.5 to pH 5.5 inthe presence of yeast. In one aspect, the total quantity of enzymeprotein required is not greater than 50 grams/ton of corn, e.g., inalternative embodiments 0.05% w/w, or 0.005% w/w, or anywhere in therange of between 0.05% w/w to 0.005% w/w.

The initial reaction rates for starch hydrolysis were determined; andthe influence of pH at varying ranges, e.g., in the range of about pH3.5-7.0, and temperatures at varying ranges, e.g., in the range of about30-40° C., on activity of enzymes of the invention were studied; thebond-type specificity of exemplary amylases and/or glucoamylases of theinvention was also determined.

Methods:

1. Determination of Protein Concentration

Lyophilized supernatants of P. pastoris cultures expressingglucoamylases and alpha-amylases were suspended in water at aconcentration of ˜10 mg of powder/ml. After protein contentdetermination by the Bradford protocol, 5 μg of protein sample andstandardized BSA solution were run on a 4-20% Tris-Glycine gradient gel.The gel was scanned on a BioRad GS800™ gel scanner following Coomassieblue staining. The Bio-Rad QUANTITY ONE™ software was used for thequantification of the BSA and glucoamylase (or alpha-amylases) bands,and the actual enzyme concentration was then calculated. Proteinconcentration was adjusted accordingly and confirmed by additional SDSPAGE.

2. Determination of Initial Reaction Rates.

Unless mentioned otherwise, assays were performed in triplicate at 37°C. and pH 5.0 in buffer (50 mM NaCH₃CO₂, 10 mM CaCl₂; 10 mM NaN₃ and0.01% Triton X-100) containing 1% raw starch, or 0.5% dextrin or 1%“soluble corn starch”. Assays were performed at 0.5 ml scale forglucoamylase and 0.25 ml scale for alpha-amylase (alpha-amylase) in anEppendorf tabletop incubator with constant shaking (800 rpm).

For glucoamylases, reactions were started by adding the enzyme (finalconcentration 0.25 μg/ml) to the reaction mix. At 0, 2.5, 5, 7.5, 10,15, 20 and 30 min, 50 μl aliquots of the reactions were withdrawn andquenched by addition to 100 μl of 1M Tris buffer, pH 7.5. Foralpha-amylases (alpha-amylases), reactions were started by adding theenzyme (final concentration 0.4 μg total protein/ml for SEQ ID NO:56,13434 and SEQ ID NO:52; 2 μg/ml for SEQ ID NO:62; 4 μg/ml for SEQ IDNO:70) to the reaction mix, and 10 μl aliquots of reactions werewithdrawn and quenched in BCA reagent at 2, 5, 10, 15, 20, 25, 30, and40 min. For determination of temperature profiles, assays were performedat 30, 34, 37 and 40° C.

The effect of pH on glucoamylase and amylase activities was evaluated atpH 3.5, 4, 5, 6 and 7, using the broad pH range Britton-Robinson buffer(50 mM CH₃COOH; H₃PO₄; H₃BO₃). Parallel reactions at pH 4, 5 and 6 werealso performed in the presence of 50 mM acetate buffer to ensure thatthe buffer used did not influence the results. For the determination ofpH profile of two calcium-dependent alpha-amylases (alpha-amylases) SEQID NO:56 and SEQ ID NO:62, malic acid/acetate/IVIES buffers were usedinstead of Britton-Robinson.

Preparation of “soluble corn starch” for reaction with alpha-amylases.Dextrin (Sigma D2006) could not be used as a substrate in the BCAalpha-amylase reactions due to the high reducing ends background.Therefore a heated corn starch (Syngenta material) was employed as asubstrate. 2% corn starch was dissolved in deionized water and heatedwith mixing in a boiling water bath for 30-40 minutes, until the starchhad dissolved and the solution appeared milky, but translucent. Thesolution of heated starch was used for 2 days, after which time somesigns of retrogradation were observed (appearance of starch clumps), andthe solution was discarded.

3. Glucose Oxidase/Peroxidase (GO) Assay for the Quantification ofGlucose Released During Starch Hydrolysis.

A coupled glucose oxidase/peroxidase (GO) assay was used to determinethe amount of glucose released by glucoamylase during starch hydrolysis.GO reactions were started by adding 10 μl of the quenched starchhydrolysis reaction to 90 μl of PBS containing glucose oxidase (0.1U/ml), peroxidase (0.25 U/ml) and 0.05 mM Amplex Red, in black Nunc96-well plates. Plates were kept at room temperature in the dark for 30min prior to reading on a fluorescent plate reader with Ex/Em 545/590nm. A standard curve with glucose concentrations of 0 to 100 μM was usedto assess the amount of glucose produced in the hydrolysis reactions.Initial rates of starch hydrolysis (nmols of glucose released from 1%granular starch/min/μg glucoamylase) were determined by plotting theamount of glucose released over time, and calculating the slope of thebest linear fit through the data points.

4. BCA Assay for Determining the Increase in Concentration of ReducingEnds During Starch Hydrolysis.

A 10 μl aliquot of amylase starch hydrolysis reaction was quenched into100 μl of BCA reagent (consisting of 64 mg/mL sodium carbonatemonohydrate, 24 mg/mL sodium bicarbonate, 1.95 mg/mL BCA, 1.24 mg/mLcupric sulfate pentahydrate, 1.26 mg/mL L-serine). Color developmentoccurred during incubation of the quenched reaction at 80° C. for 35minutes and was followed by absorbance determination at 560 nm. Initialrates were calculated over a 40 min reaction time. A standard curveusing maltose (0-54 μM) was constructed to correlate A_(560 nm) with theconcentration of generated reducing sugars (nmoles). Specific activitywas expressed as nmoles/min/μg enzyme.

5. Bond-Type Specificity of Glucoamylases with Maltose and Isomaltose asSubstrates.

Reactions were started by adding the enzyme (final concentration 5 μg/mlfor maltose, and 30 μg/ml for isomaltose) to the reaction mix. At 2, 5,10, 15, 20, 25, 30 and 40 min, 5 μl aliquots of the reactions werewithdrawn and quenched by addition to 10 μl of 1M Tris buffer, pH 7.5.Nine substrate concentrations were used in the studies, ranging from 0to 12 mM for maltose and 2.5 to 120 mM for isomaltose. The reactionswere performed in triplicate at 37° C. and pH 5.0 in buffer (50 mMNaCH₃CO₂, 10 mM CaCl₂), at 50 ul scale in an Eppendorf tabletopincubator with constant shaking (800 rpm). Glucose production wasmeasured at the end of the reaction using the glucose oxidase/peroxidase(GO) assay.

Results

1. Characterization of Glucoamylases:

1.1 Initial Reaction Rates:

Initial rates for granular and soluble starch hydrolysis are presentedin Table 2, illustrated in FIG. 24. As can be seen from Table 2,amylases and/or glucoamylases of the invention displayed up to 3× betteractivity (SEQ ID NO:48) against granular starch, with similar orslightly better activity on soluble starch when compared to thebenchmark A. niger enzyme. SEQ ID NO:20 did not appear to display anyactivity against granular starch under the conditions tested (probablydue to the lack of a Starch Binding Domain). Table 2 (FIG. 24)summarizes data comparing initial rates of granular corn starch andsoluble starch (dextrin) hydrolysis by exemplary enzymes of theinvention (including those with glucoamylases activity) and a benchmarkenzyme A. niger glucoamylase at 37° C., pH 5.0; initial rates areexpressed as nmols of glucose/min/μg of glucoamylase protein releasedfrom 1% granular starch or from 0.5% dextrin. Each number is the averagevalue from 6-10 data points.

1.2 Temperature Profile:

The effect of temperature on the activity of exemplary enzymes of theinvention, including glucoamylases, on granular starch as a substratewas determined; a “benchmark” enzyme, the commercially available A.niger glucoamylase, was used. Glucose release was measured 30, 32, 34,36, 38 and 40° C. at pH 5.0. Activities of glucoamylases increased withtemperature; they were most active at 40° C. but retained approximately50% of peak activity at 30° C.

FIG. 25 illustrates the effect of temperature on the activity of theexemplary glucoamylase SEQ ID NO:20 and the A. niger “benchmark” withsoluble starch (dextrin) as a substrate. Glucose release was measured atthe indicated temperature at pH 5.0.

1.3 pH Profile:

The influence of pH on starch hydrolysis was tested with both granularand soluble starch, respectively. All glucoamylases hydrolyzed bothsubstrates best at lower pH, with the exemplary SEQ ID NO:26 being themost acidic in character. For both substrates, glucose release wasmeasured at the indicated pH at 37° C. Initial rates were calculatedover 20 min and converted to a percentage of the maximum rate.

1.4 Bond-Type Cleavage Specificity:

The kinetic parameters for the hydrolysis of maltose (alpha-1,4-linkage)and isomaltose (alpha-1,6-linkage) were determined for 7 selectedglucoamylases and the “benchmark” A. niger glucoamylase. The experimentswere conducted with lyophilized P. pastoris lysates and the proteinswere not purified; therefore only data independent of proteinconcentration are reported herein. Table 3, below, summarizes values ofK_(M) for maltose and isomaltose and the ratio of k_(cat)/K_(M) formaltose compared to k_(cat)/K_(M) for isomaltose. These parametersdetermined for A. niger glucoamylase are in good agreement withpublished data; e.g., K_(M) for maltose is reported to be 1.2-2.1 mM;K_(M) for isomaltose is reported to be 19.8-42.0 and k_(cat)/K_(M) formaltose over k_(cat)/K_(M) for isomaltose is reported to be between300-600, according to Frandesen (1995) Biochemistry. 34:10162-10169;Sierks and Svensson (1996) Biochemistry 35:1865-1871; Fagerstrom andKalkkinen (1995) Biotechnol. Appl. Biochem. 21:223-231.

Table 3, summarizes the kinetic parameters for hydrolysis of maltose andisomaltose by seven exemplary glucoamylases of this invention and a“benchmark” A. niger glucoamylases; each number is the average valuefrom 5 different experiments:

TABLE 3 Maltose Isomaltose k_(cat)/K_(M) (maltose)/ K_(M) (mM) K_(M)(mM) k_(cat)/K_(M) (isomaltose) SEQ ID NO: 28 0.61 ± 0.06 11.94 ± 4.99750 SEQ ID NO: 74 1.87 ± 0.17 11.55 ± 2.92 481 SEQ ID NO: 20 2.62 ± 0.19 53.97 ± 23.17 897 SEQ ID NO: 14 2.67 ± 0.15  41.5 ± 5.05 116 SEQ ID NO:26 0.98 ± 0.33 12.18 ± 0.64 456 SEQ ID NO: 48 2.26 ± 0.12 21.69 ± 3  565 SEQ ID NO: 18 1.01 ± 0.09 11.74 ± 7.74 415 A. niger 0.93 ± 0.1 18.72 ± 3.95 249 glucoamylase

As can be seen from Table 3, the exemplary glucoamylase SEQ ID NO:20 wasmost strongly selective for maltose and had nearly 900-fold higherspecificity towards alpha-1,4-linkages relative to alpha-1,6-bonds. Theleast selective glucoamylase was SEQ ID NO:14 with ˜100-fold higherspecificity towards alpha-1,4-bonds relative to alpha-1,6-bonds.

Example 24: Characterization of Alpha-Amylases and/or Glucoamylases ofthe Invention

This example describes the characterization of alpha-Amylases and/orglucoamylases of the invention.

Initial Reaction Rates:

The initial rates of hydrolysis of granular and soluble starch by eight(8) exemplary alpha-amylases were compared with a “benchmark”alpha-amylase from A. oryzae, and the results are summarized in Table 4,below. Due to the relatively high background seen in BCA assays with A.oryzae alpha-amylase from Sigma (A6211), a second preparation of thesame amylase from Megazyme (E-ANAAM™) was also evaluated. Resultsobtained with both preparations were very similar. As can be seen fromTable 34, all amylases tested displayed significantly higher activityagainst soluble starch when compared with granular starch. However, thisdifference was less marked for the exemplary Amylases and/orglucoamylases of the invention than for the “benchmark” A. oryzaeenzyme.

Table 4 shows data comparing initial rates of hydrolysis of granularcorn starch and soluble corn starch by eight (8) exemplary α-Amylasesand/or glucoamylases of the invention and the “benchmark” alpha-amylasefrom A. oryzae at 37° C. and pH 5 (note: *Initial rates are expressed asnmole of reducing ends released from 1% starch/min/μg of alpha-amylaseprotein for SEQ ID NO:56, SEQ ID NO:62, SEQ ID NO:2, and SEQ ID NO:52;**Initial rates for SEQ ID NO:70, SEQ ID NO:66 and SEQ ID NO:76 areexpressed as nmoles of reducing ends released from 1% starch/min/μg oftotal protein; each number is an average value from 5 data points:

TABLE 4 Initial rate* ± SD Initial rate* ± SD Enzyme Granular starchSoluble starch SEQ ID NO: 56 15.7 ± 1.67  1607.9 ± 518.22 SEQ ID NO:70** 2.1 ± 0.24 28.4 ± 3.81 SEQ ID NO: 62 3.5 ± 0.37 139.6 ± 55.96 SEQID NO: 66** 0.8 ± 0.09  81.3 ± 31.11 SEQ ID NO: 2   7 ± 0.75 381.2 ±74.15 SEQ ID NO: 52 10.3 ± 1.75  248.2 ± 28.46 SEQ ID NO: 76** 2.6 ±0.41 160.3 ± 42.62 A. oryzae amylase 0.4 ± 0.07 498.7 ± 64.78(MegazymeE-ANAAM)

Temperature Profile:

The effect of temperature on starch hydrolysis by the characterizedα-amylases was determined. Activity was measured at pH 5.0 during 40 minincubation at the indicated temperature, and initial rates werecalculated and plotted against time. Activities of amylases wereaffected by temperature to a different degree. Five of the exemplaryAmylases and/or glucoamylases of the invention SEQ ID NO:56, SEQ IDNO:70, SEQ ID NO:76 and SEQ ID NO:66, were most active at 40° C. andretained approximately 30% of activity at 30° C. Activities of SEQ IDNO:2, SEQ ID NO:52 and SEQ ID NO:62 were only marginally affected bychanges in temperature over the range investigated.

pH Profile:

The influence of pH on exemplary Amylases and/or glucoamylases of theinvention on starch hydrolysis was tested with both granular and solublestarch substrates, and the results were determined. The increase inreducing ends was measured at the indicated pH at 37° C. Initial rateswere calculated over 40 min and converted to a percentage of the maximumrate. The exemplary SEQ ID NO:52 (originally of fungal origin) had thelowest pH optimum (approximately pH 4). Another exemplary enzyme SEQ IDNO:2 (also originally of fungal origin) also displayed preference foracidic pH, with an apparent optimum at approximately pH 4.5-5. Theexemplary amylase SEQ ID NO:66 (originally of Archaeal origin) had anapparent optimum of approximately pH 5.0, retaining approximately 70% ofpeak activity at pH 4.0. The remaining exemplary enzymes of theinvention had apparent optima between pH 5.0 and 6.0, and were almostinactive at pH 4.0 and 3.5.

Example 25: Expression of Amylases and/or Glucoamylases of the Inventionin Host Cells

This example describes the evaluation of expression of exemplary enzymesof the invention in different hosts cells; in particular, the expressionof three (3) exemplary amylases in different expression hosts: Pichiapastoris, Hansenula polymorpha, and Cochliobolus heterostrophus werestudied.

H. polymorpha: Two expression vectors (Artes Biotechnology GmbH,Erkrath, Germany) were used: pFPMT-Mfa™ with a formate dehydrogenasepromoter and pTPS1-Mfa™ with a trehalose-6-phosphate synthase promoter.In both vectors the secretion signal used was the pre-prosequence of themating-factor MFa1 from Saccharomyces cerevisiae.

Plasmids with subcloned amylase genes were introduced into H. polymorphacompetent cells by electroporation. 96 colonies from each transformationwere selected and grown for approximately 30 (3 passages) to 80 (8passages) generations by various sub-culturing steps, under selectiveconditions in liquid medium (YNB-Glucose). These steps allow forincreasing the copy number and facilitate the integration of the plasmidinto the chromosome. After these 3 or 8 passaging steps, transformantswere cultivated under non-selective conditions (YPD medium) to test forplasmid stabilization (loss of any non-integrated plasmid). Screeningfor positive clones was performed on Red Starch-agar plates. Clones withlarger than the control (same gene expressed in P. pastoris) clearingzone were selected as primary hits. Amylase expression by the selectedclones was confirmed by assaying culture supernatants on BODIPY-starchsubstrate, and SDS PAGE was used for visualization of the proteinproduced. 1 L cultures were prepared for further testing.

Cochliobolus heterostrophus. cDNA and gDNA of amylase from C.heterostrophus were subcloned into pCh-ubi (ubiquitin promoter) andpCh-GPD (GPD promoter) and targeted to the PKS18 locus of the C5 strainof C. heterostrophus by PEG-mediated protoplast transformation, usinglinearized construct DNA. One of the constructs, a genomic version underan ubi (ubiquitin) promoter, was also introduced into the JMD3 strainfrom which the amylase-encoding gene had been deleted. Similarly, as inthe case of P. pastoris, screening of the transformants was performed onred starch plates and confirmed by BODIPY-starch assays and SDS PAGEwith culture supernatants. No difference was observed in expressionbetween the cDNA and the genomic versions. Similarly no significantdifference was observed with the two different promoters.

Results:

Strains of: H. polymorpha and C. heterostrophus expressing, forcomparison, 3 exemplary amylase-encoding and glucoamylase-encoding geneswere constructed, in addition to 3 original P. pastoris strains. Allstrains used for comparisons and preliminary fermentation yieldsobtained with these strains are presented in Table 5, summary estimatedexpression yields (g/L) of exemplary Amylases and/or glucoamylases ofthe invention produced in Pichia:

TABLE 5 Origin of the amylase gene Pichia pastoris fungal SEQ ID NO: 20.01* bacterial SEQ ID NO: 70 0.07* archaeal SEQ ID NO: 66 0.13**fermentation yieldFollowing the expression evaluation, protein purity, specific activityand performance in granular starch hydrolysis assays were compared.These comparisons led to the following conclusions:

-   -   No significant biochemical differences were observed between        enzymes expressed in the different hosts.    -   Expression in Hansenula did not appear to result in an        improvement over the expression observed in P. pastoris.        Furthermore, the expressed proteins appeared to be highly        glycosylated.

Example 26: Raw Starch Fermentation Using Amylases of this Invention

This example describes exemplary methods for raw starch fermentationusing amylases of this invention.

Fifteen (15) exemplary enzymes consisting of eight (8) alpha-amylasesand seven (7) glucoamylases were initially identified. The “lead”exemplary enzymes were determined based on activity at temperatures from30° to 40° C., pH range from 3.5-7.0, and hydrolysis of 1% raw starch.Unpurified microbial enzyme was first analyzed for amount of proteinsand expression ratios; and then evaluated in raw starch fermentations.The total enzyme loading and ratios of alpha-amylases to glucoamylaseswere then determined.

Note: for the raw starch fermentation work, all enzymes were obtained byexpression in Pichia pastoris, except for SEQ ID NO:78 (encoded, e.g.,by SEQ ID NO:77), which was expressed in Pseudomonas fluorescens (e.g.,as described in JBC, 2002, 277(29):26501-26507).

TABLE 1.1 Enzyme Expression and Relative Purity of “lead” exemplaryalpha-amylases and glucoamylases protein/powder Expression Enzyme***(g/g)* ratio** SEQ ID NO: 56 0.12 ± 0.42  0.6 SEQ ID NO: 52 0.09 ± 0.0030.6 SEQ ID NO: 66 0.05 ± 0.005  0.1 (0.5****) SEQ ID NO: 66 0.05   0.1(0.5****) SEQ ID NO: 66 0.059 ± 0.002  0.5**** SEQ ID NO: 66 0.0850.5**** SEQ ID NO: 2 0.03 ± 0.005 0.3 SEQ ID NO: 2 0.03 ± 0.002 0.3 SEQID NO: 70 0.09 ± 0.003 0.05 (0.25****) SEQ ID NO: 70 0.126 ± 0.002 0.25**** SEQ ID NO: 70 0.131 0.25**** SEQ ID NO: 62 0.2 ± 0.03 0.75 SEQID NO: 78 10 (mgs/ml) 0.25 SEQ ID NO: 76 0.1 ± 0.01 0.2 SEQ ID NO: 26 0.2 ± 0.026 0.8 SEQ ID NO: 28 0.15 ± 0.076 0.45 SEQ ID NO: 28 0.36 ±0.042 0.45 SEQ ID NO: 18 0.24 ± 0.096 0.8 SEQ ID NO: 74 0.21 ± 0.016 0.8SEQ ID NO: 48 0.28 ± 0.098 0.5 SEQ ID NO: 20 0.28 ± 0.066 0.7 SEQ ID NO:14 0.21 ± 0.155 0.2 Control (empty vector)  0.0063 *protein content wasdetermined using Bradford's protocol. **expressed as percentage of totalprotein in clarified lysate; determined by SDS PAGE using 5 μg proteinloadings, and the Bio-Rad Quantity One software. ***where an enzymeappears several times in this list, different lots were used. ****valueafter de-glycosylation.

Experimental Methods

Raw Starch Fermentation

All fermentations were done using Yellow Dent II corn, milled on theUltra Centrifugal Mill by Glen Mills at 12,000 rpm through a 0.5 mmscreen. The corn flour was analyzed on a Mettler HB-43™ moisture balanceto determine moisture content and then 10 g dry weight of corn flour wasmeasured into a 50 ml flask with stir bar. Water was then added followedby the addition of tetracycline (0.5 mg), H2SO4 (175 μl of 0.9 Msolution), unpurified/purified enzyme stocks, and yeast to bring thetotal slurry to 33% total solids. The flasks were placed on a stir platein 30° C. for 72 hours. Samples were taken at 50 or 72 hours offermentation for analyses. Sigma Aspergillus niger glucoamylase (A-7095)was used as the benchmark added at 2.065 Units (approximately 0.707 mgtotal protein, or 0.07% total protein loading)/g dw of flour and thetest enzyme total loading (%) is based on the total weight of expressedenzyme (g)/dry weight of corn flour in fermentation (g).

To analyze ethanol content and sugar profiles, 1.5 ml of fermentationslurry was taken from each flask at 50 or 72 hours, placed in a 1.8 mlmicrofuge tube, and spun down at 13000 rpm for 5 minutes. Thesupernatant was poured into a 0.45 μm spin-x column, spun down at 7000rpm for 5 minutes, and 200 μl of the spin through was aliquoted into anHPLC vial and analyzed by HPLC. A Waters HPLC equipped with a refractiveindex detector was used. The column used was the Bio-Rad AMINEX HPX-87H™(Cat. #: 125-0140). The mobile phase used was 0.005 M H₂SO4 at 0.6mL/minute.

Preparation of Standards

The standards used for the analysis described herein contain glucose,maltose, maltotriose, maltodextrin, fructose, glycerol, lactic acid,acetic acid, succinic acid, ethanol and methanol. One composite standardis prepared and subsequently diluted to produce the varyingconcentrations needed to establish standard curves for each compound.All reagents used for standard preparation should be stored in adessicator cabinet, refrigerated or stored per manufacturers'recommendations. The most accurate means of measuring the componentsshould be employed. Into a clean dry 100 mL volumetric flask add all dryreagents.

-   -   Add ˜35 mL water and swirl to dissolve solids.    -   Place a 50 mL Falcon tube on the balance add 25 mL water and        then tare.    -   To the water add glycerol, ethanol, methanol, lactic and acetic        acids.    -   Gently vortex to mix and add contents to volumetric flask with        sugar solution.

Rinse 50 mL Falcon tube 3 times with ˜5 mL water each time and add rinseto volumetric flask.

-   -   Bring entire volume of flask to 100 mL. This solution represents        the mixed 20% ethanol standard. The solution is sterilized using        a Millipore STERIFLIP™ 0.22 or 0.45 μm filter.    -   Dilutions of this standard are used to create the mixed 5, 10,        and 15% ethanol standards.    -   A mixed 5% standard consists of 1 part mixed 20% standard and 3        parts water.    -   A mixed 10% standard is a simple 1:1 dilution of the mixed 20%        standard.    -   A mixture of 3 parts mixed 20% standard and 1 part water will        create the mixed 15% standard.    -   A certified 10.3% v/v ethanol standard (Sigma Cat. # E2385)        should be run each time a new standard is prepared or monthly to        verify the accuracy of the Mixed standard set.        Results

Several alpha-amylases and glucoamylases were tested in combinations inraw starch fermentations at different total enzyme loadings of 0.001,0.01, and 0.1% (w/w). The alpha-amylases tested were SEQ ID NO:56, SEQID NO:70, SEQ ID NO:52, SEQ ID NO:2, SEQ ID NO:62, and SEQ ID NO:66, SEQID NO:76. The glucoamylases tested were SEQ ID NO:48, SEQ ID NO:26, SEQID NO:18, SEQ ID NO:74, SEQ ID NO:28, SEQ ID NO:14, and SEQ ID NO:20.All combinations were given an ID to be used in the experimentalprocess. Total enzyme loading was calculated based on the expressionratio information provided herein. The alpha-amylase to glucoamylaseratio was determined based on previous data. Ethanol yield was measuredafter 50 or 72 hours of fermentation by HPLC analysis. Results indicatedthat all enzyme combinations produced various levels of ethanol and somecombinations outperformed the Sigma GA benchmark (Table 1.2).

TABLE 1.2 Ethanol yield in raw starch fermentations of “lead” exemplaryalpha- Amylases and Glucoamylases after 50 hours of fermentation^(a).Ethanol Yield (% v/v) 0.001% 0.01% 0.1% total total total Alpha-amylaseglucoamylase AA:GA enzyme enzyme enzyme (AA) (GA) ratio loading loadingloading Combo 1 SEQ ID NO: 56 SEQ ID NO: 26 2:1 6.12 11.60 15.56 Combo 2SEQ ID NO: 56 SEQ ID NO: 18 1:2 6.97 12.53 15.69 Combo 3 SEQ ID NO: 56SEQ ID NO: 28 1:2 5.96 11.06 16.42 Combo 4 SEQ ID NO: 56 SEQ ID NO: 741:2 6.83 11.49 14.29 Combo 5 SEQ ID NO: 56 SEQ ID NO: 20 1:2 4.28 5.617.55 Combo 6 SEQ ID NO: 56 SEQ ID NO: 48 1:2 6.40 12.58 15.86 Combo 7SEQ ID NO: 66 SEQ ID NO: 26 1:2 7.39 15.38 N/A Combo 8 SEQ ID NO: 66 SEQID NO: 18 1:2 5.84 12.81 N/A Combo 9 SEQ ID NO: 66 SEQ ID NO: 28 1:26.12 12.62 N/A Combo 10 SEQ ID NO: 66 SEQ ID NO: 74 1:2 7.02 12.29 N/ACombo 11 SEQ ID NO: 66 SEQ ID NO: 20 1:2 4.48 5.72 N/A Combo 12 SEQ IDNO: 66 SEQ ID NO: 48 1:2 6.54 14.16 N/A Combo 13 SEQ ID NO: 70 SEQ IDNO: 26 1:2 9.08 15.04 16.76 Combo 14 SEQ ID NO: 70 SEQ ID NO: 18 1:27.86 13.00 14.71 Combo 15 SEQ ID NO: 70 SEQ ID NO: 74 1:2 8.18 13.2716.40 Combo 16 SEQ ID NO: 70 SEQ ID NO: 48 1:2 8.36 13.80 15.90 Combo 17SEQ ID NO: 52 SEQ ID NO: 26 1:2 7.92 12.67 15.15 Combo 18 SEQ ID NO: 52SEQ ID NO: 18 2:1 10.66 16.10 14.92 Combo 19 SEQ ID NO: 52 SEQ ID NO: 481:2 10.88 15.97 14.87 Combo 20 SEQ ID NO: 62 SEQ ID NO: 26 1:2 7.3012.67 15.68 Combo 21 SEQ ID NO: 62 SEQ ID NO: 48 1:2 7.24 11.13 14.84Combo 22 SEQ ID NO: 2 SEQ ID NO: 26 2:1 9.20 14.56 15.36 Combo 23 SEQ IDNO: 2 SEQ ID NO: 48 1:2 9.81 15.42 15.61 Combo 24 SEQ ID NO: 56 SEQ IDNO: 14 1:2 5.46 7.86 13.67 Combo 25 SEQ ID NO: 70 SEQ ID NO: 14 2:1 6.9910.85 15.72 Combo 26 SEQ ID NO: 78 SEQ ID NO: 48 1:2 N/A N/A 16.13^(a)Combo 27 SEQ ID NO: 78 SEQ ID NO: 26 1:2 N/A N/A 16.88^(a) Combo 28 SEQID NO: 76 SEQ ID NO: 48 1:2 N/A N/A 17.15^(a) Sigma GA N/A N/A 16.82^(b)control ^(a)Ethanol yield after 72 hours of fermentation. ^(b)Totalenzyme loading was estimated to be approximately 0.07%.

Example 27: Expression of Enzymes in Plants; Raw Starch Fermentation

This example describes plant expression of nine exemplary enzymes ofthis invention (enzymes not having codon optimization), and raw starchfermentation using plant material.

Five (5) alpha-amylases, SEQ ID NO:52, SEQ ID NO:4, SEQ ID NO:70, SEQ IDNO:66, and SEQ ID NO:76 and 4 glucoamylases, SEQ ID NO:48, SEQ ID NO:18,SEQ ID NO:26, and SEQ ID NO:28 were chosen for plant expression based onhigh ethanol yield of the microbial enzymes in raw starch fermentation.This maize expression was carried out with the gene sequences withoutcodon optimization. The objectives of this study were to evaluate thepotential expression levels of these enzymes and to evaluate whetherexpression of these enzymes would have detrimental effect on maizeagronomics through out its developmental stages.

Experimental Methods

Vector Construction

Table 2.1 summarized the abbreviations used for the DNA elements in themaps shown below.

TABLE 2.1 Abbreviations used in the construct maps Name FunctionDescription cAMY(SEQ ID CDS Alpha amylase, SEQ ID NO: 66, minus thenative NO: 66)-02 leader, attached to a Gamma Zein signal sequencetargeting to the apoplast cAmy(SEQ ID CDS Glucoamylase, SEQ ID NO: 18,minus the native NO: 18)-02 leader, attached to a Gamma Zein signalsequence targeting to the apoplast cAmy(SEQ ID CDS Alpha amylase, SEQ IDNO: 52, minus the native NO: 52)-02 leader, attached to a Gamma Zeinsignal sequence targeting to the apoplast cAmy(SEQ ID CDS Glucoamylase,SEQ ID NO: 28, minus the native NO: 28)-02 leader, attached to a GammaZein signal sequence targeting to the apoplast cAmy(SEQ ID CDSGlucoamylase, SEQ ID NO: 26, minus the native NO: 26)-02 leader,attached to a Gamma Zein signal sequence targeting to the apoplastcAmy(SEQ ID CDS Glucoamylase, SEQ ID NO: 48, minus the native NO: 48)-02leader, attached to a Gamma Zein signal sequence targeting to theapoplast cAmy(SEQ ID CDS Alpha-amylase, SEQ ID NO: 76, minus the nativeNO: 76)-02 leader, attached to the gamma zein signal sequence, targetingto the apoplast cAmy(SEQ ID CDS Alpha-amylase, SEQ ID NO: 70, minus thenative NO: 70)-02 leader, attached to a Gamma Zein signal sequencetargeting to the apoplast

The exemplary vector designated “15745,” is a binary vector containingan alpha amylase, the exemplary SEQ ID NO:70 minus its native leader.The native leader was replaced with the Gamma Zein signal sequence fortargeting to the apoplast. The Gamma Zein-SEQ ID NO:70 fusion isexpressed using the Gamma Zein promoter. The binary vector also containsa Ubi-PMI-Nos cassette for mannose selection.

A fragment of cAmy (SEQ ID NO:70)-01, containing amino acids 86-525(plus stop codon), was PCR amplified from plasmid 15649. The PCRamplification introduced an AfeI site at the 5′ end and a BglII site atthe 3′ end of the fragment. The addition of the AfeI site added analanine amino acid in front of the cAmy (SEQ ID NO:70) fragment (SY1709:92-93). This PCR amplicon was cloned into pCR2.1-TOPO and sequenced toverify the presence of the new restriction enzyme sites(SY1709:111-112,122-124). Once verified, cAmy (SEQ ID NO:70) wasdigested out of the TOPO backbone via AfeI/BglII. Similarly, construct15460ZeinAmyVN was digested with AfeI/BglII. This backbone and cAmy (SEQID NO:70) were ligated together. When ligated, cAmy (SEQ ID NO:70)stayed in frame with the signal peptide xGZein27ss-01, with only theaddition of an alanine residue at the cloning junction. Transformantswere screened for the presence of the amylase gene using primersZeinAmy1199F and prGTL-03R. Positive clones produced an amplicon of ˜1.6kB (SY1709:130). Five clones were selected and were digested with SanDIand RsrII to remove prGZein-01:cAmy (SEQ ID NO:70):t35s-08. Construct15468 was linearized with RsrII. The binary backbone and digestedcassette from one of the positive clones (SanDI/RsrII) were ligatedtogether at the RsrII restriction site. Following transformation intoTop10 cells, transformants were screened for the presence of thecassette using primers ZeinAmy1199F and Mubi-5 (SY1709:146,150). Threepositive transformants were selected and checked via a BamHI diagnosticdigest. These clones were sequenced (SY1709:162; both cloning junctionsas well as the entire cAmy (SEQ ID NO:70) coding sequence), it wasconfirmed that all three were correct in sequence.

The exemplary vector designated “15750,” is a binary vector containing aglucoamylase, the exemplary SEQ ID NO:18, minus its native leader. Thenative leader was replaced with the Gamma Zein signal sequence fortargeting to the apoplast. The Gamma Zein-SEQ ID NO:18 fusion isexpressed using the Gamma Zein promoter. The binary vector also containsan Ubi-PMI-Nos cassette for mannose selection.

A fragment of cAmy (SEQ ID NO:18)-01, containing amino acids 88-712(plus stop codon), was PCR amplified from plasmid 15652. The PCRamplification introduced an AfeI site at the 5′ end and a BglII site atthe 3′ end of the fragment. The addition of the AfeI site resulted inamino acid 88 changing from a serine to an alanine (SY1709:113-114,116). This PCR amplicon was cloned into pCR2.1-TOPO andsequenced to verify the presence of the new restriction enzyme sites(SY1709:116,131). Once verified, the cAmy (SEQ ID NO:18) fragment wasdigested out of the TOPO backbone via AfeI/BglII. Similarly, construct15460ZeinAmyVN was digested with AfeI/BglII. This backbone and the cAmy(SEQ ID NO:18) fragment were ligated together. When ligated, cAmy (SEQID NO:18) stayed in frame with the signal peptide xGZein27ss-01, withthe addition of an alanine residue at the cloning junction. Six cloneswere selected and were digested with SanDI and RsrII to removeprGZein-01:cAmy (SEQ ID NO:18):t35s-08. Construct 15468 was linearizedwith RsrII. The binary backbone and digested cassette from one of thepositive clones (SanDI/RsrII) were ligated together at the RsrIIrestriction site. Following transformation into Top10 cells,transformants were screened for the presence of the cassette usingprimers ZeinAmy1199F and Mubi-5 (SY1709:181). Additionally, all 6transformants were selected and checked via a SacI diagnostic digest.Three of the positive clones were sequenced (SY1709:189-190) (bothcloning junctions as well as the entire cAmy (SEQ ID NO:18) codingsequence), it was confirmed that all three were correct in sequence.

The exemplary vector designated “15751,” is a binary vector containing aglucoamylase, the exemplary SEQ ID NO:48, minus its native leader. Thenative leader was replaced with the Gamma Zein signal sequence fortargeting to the apoplast. The Gamma Zein-SEQ ID NO:48 fusion isexpressed using the Gamma Zein promoter. The binary vector also containsa Ubi-PMI-Nos cassette for mannose selection.

Using 15651 as a template, mutagenesis primers were used in a multi-sitemutagenesis reaction (QUICKCHANGE™, Stratagene) to remove an internalAfeI site, introduce an AfeI site at the 5′ end (mutation was in signalsequence, no peptide change), and introduce an external 3′ BglII sitefor cloning. Restriction enzyme analysis was performed and clones thatwere positive for having the internal AfeI site removed and the BglIIsite introduced were selected. A positive clone was sent for sequenceanalysis which was confirmed. This clone DNA was then used as templatefor PCR amplification to introduce an AfeI site at the 5′ end of theglucoamylase domain. The mutation was in the native signal sequence anddid not change the glucoamylase peptide sequence. The PCR product wasdigested sequentially with AfeI and then with BglII. The digestedproduct was gel purified and ligated to the 15460AmyZeinVN backbone,creating cAmy (SEQ ID NO:48) with a gamma zein signal sequence targetingto the apoplast. This intermediate vector was sequence verified and thendigested with SanDI and RsrII sequentially. This fragment was gelpurified and then ligated into 15468 binary cut with RsrII and CIPtreated. The positive clones were PCR size screened and then one of thepositive clones was sent off for sequence analysis of the entirefragment ligated into the binary. Sequence was confirmed.

The exemplary vector designated “15761,” is a binary vector containingan alpha amylase, the exemplary SEQ ID NO: 66, minus its native leader.The native leader was replaced with the Gamma Zein signal sequence fortargeting to the apoplast. The Gamma Zein-SEQ ID NO:66 fusion isexpressed using the Gamma Zein promoter. The binary vector also containsa Ubi-PMI-Nos cassette for mannose selection.

A fragment of cAmy (SEQ ID NO:66)-01, containing amino acids 86-521 wasmutagenized using site directed mutagenesis according to the StratageneQuick Change protocol. The mutagenesis required 3 individual primers(all amplifying in the forward direction) and introduced threemutations: an AfeI site at the 5′ end, a BglII site at the 3′ end(external to the gene), and the removal of an internal AfeI site(SY1709:174). The mutagenized construct was transformed into Top10 cellsfor screening to determine if any/all of the desired mutations werepresent. 15 transformants were selected for screening and were digestedseparately with AfeI and BglII (SY1709:185-186). It was determined fromthe restriction digests that there were 6 possible clones with all threeof the desired mutations; three of these clones were sent for sequencing(SY1709:190; SY1777:11). For all three clones, an AfeI site had beenintroduced at the 5′ end and the internal site had been removed. Inaddition, the 3′ BglII site had been introduced. Following confirmation,the alpha amylase was digested out of the mutagenized construct withAfeI/BglII and ligated into the 15460ZeinAmyVN backbone which had alsobeen digested with AfeI and BglII. When ligated, cAmy (SEQ ID NO:66)stayed in frame with the signal peptide xGZein27ss with no amino acidchanges at the cloning junction. Transformants were screened for thepresence of the amylase gene using primers ZeinAmy1199F and prGTL-03R.Positive clones produced an amplicon of ˜1.6 kB (SY1777:24). Threeclones were selected and digested with SanDI/RsrII to removeprGZein-01:cAmy (SEQ ID NO:66):t35s-08. Construct 15468 was linearizedwith RsrII. The binary backbone and digested cassette from one of thepositive clones (SanDI/RsrII) were ligated together at the RsrIIrestriction site. Following transformation into Top10 cells,transformants were screened for the presence of the cassette usingprimers ZeinAmy1199F and prGTL-03R (SY1777:47-48). Six positivetransformants were selected and checked via a HindIII diagnostic digest.Three of these clones were sequenced (SY1777:56, 62) at both cloningjunctions as well as the entire coding sequence; it was confirmed thatall three were correct in sequence.

The exemplary vector designated “15756,” is a binary vector containing aglucoamylase, the exemplary SEQ ID NO:26, minus its native leader. Thenative leader was replaced with the Gamma Zein signal sequence fortargeting to the apoplast. The Gamma Zein-SEQ ID NO:26 fusion isexpressed using the Gamma Zein promoter. The binary vector also containsa Ubi-PMI-Nos cassette for mannose selection.

Using exemplary vector 15653 as a template, mutagenesis primers wereused in a multi-site mutagenesis reaction (QUICKCHANGE™, Stratagene) toremove an internal AfeI site, introduce an AfeI site at the 5′ end(mutation was in signal sequence, no peptide change), and introduce anexternal 3′ SacI site for cloning. The reaction was transformed intoTop10 competent cells. Colonies were picked and screened via restrictionenzyme analysis. Clones that were positive for having the internal AfeIsite removed, the 5′ Afe site inserted and the SacI site introduced wereselected. A positive clone was sent off for sequence analysis which wasconfirmed. This clone DNA was then digested with SacI and AfeI. Thedigest was then gel purified and ligated to the 15460AmyZeinVN backbonecreating cAmy (SEQ ID NO:26) with a gamma zein signal sequence targetingto apoplast. This intermediate vector was sequence verified thendigested with SanDI and RsrII. This fragment was gel purified andligated into 15468 binary vector cut with RsrII and CIP treated. Thepositive clones were PCR size screened and then one of the positiveclones was sent off for sequence analysis of the entire fragment ligatedinto the binary. Sequence was confirmed.

The exemplary vector designated “15742,” is a binary vector harboring(comprising) the C-terminal 616 amino acids of a glucoamylase, theexemplary SEQ ID NO:28 (cAmy (SEQ ID NO:28)-01) fused in-frame with theGamma Zein signal sequence (xGZein27ss-01), targeting the glucoamylaseto the apoplast to create (cAmy (SEQ ID NO:28)-02). Expression is drivenby the 5′ region from Gamma Zein A (prGZein-01), which is a seedspecific promoter.

Vector 15654 harboring cAmy (SEQ ID NO:28)-01 served as PCR DNA templatemodified by the addition of an NcoI site (5-prime end) and a BglII site(3-prime end). The PCR product was TOPO-cloned and Clone #1 wassequenced for validation (SY1533:181). The new component alias “cAmy(SEQ ID NO:28)” was digested with NcoI/BglII and gel purified. Thefragment was ligated in-frame to the NcoI/BglII site of a cloning vectoralias “15460ZeinAmyVN” thereby generating a cassette harboringprGZein-01:xGZein27ss-01:cAmy (SEQ ID NO:28):iPEPC9-01:t35s-08(SY1533:183). The gene cassette was digested sequentially withSanDI/RsrII, gel purified and ligated into the RsrII site of binaryvector 15468. Successful ligation was confirmed by PCR and DNA sequence(SY1533:185-187).

The exemplary vector designated “15749,” is a binary vector containingan alpha amylase, the exemplary SEQ ID NO:52, minus its native leader.The native leader was replaced with the Gamma Zein signal sequence fortargeting to the apoplast (cAmy (SEQ ID NO:52)-02). The Gamma Zein-SEQID NO:52 fusion is expressed using the Gamma Zein promoter. The binaryvector also contains a Ubi-PMI-Nos cassette for mannose selection.

A fragment of cAmy (SEQ ID NO:52)-01, containing amino acids 88-674(plus stop codon), was PCR amplified from plasmid 15648. The PCRamplification introduced an AfeI site at the 5′ end and a SacI site atthe 3′ end of the fragment. The addition of the AfeI site resulted inamino acid 88 changing from a serine to an alanine (SY1709:95-96). ThePCR amplicon was cloned into pCR2.1TOPO and sequenced to verify thepresence of the new restriction enzyme sites (SY1709:103,111-112). Onceverified, cAmy (SEQ ID NO:52) was digested out of the TOPO backbone viaAfeI/SacI. Similarly, construct 15460ZeinAmyVN was digested withAfeI/SacI. This backbone and cAmy (SEQ ID NO:52) were ligated together.When ligated, cAmy (SEQ ID NO:52) stayed in frame with the signalpeptide xGZein27ss-01 with only the first amino acid residue changedfrom a leucine to an alanine. Transformants were screened for thepresence of the amylase gene using primers ZeinAmy1199F and prGTL-03R.Positive clones produced an amplicon of ˜2.1 kB (SY1709:130). Fiveclones were selected and were digested with SanDI and RsrII to removeprGZein-01:cAmy (SEQ ID NO:52):t35s08. Construct 15468 was linearizedwith RsrII. The binary backbone and digested cassette from one of thepositive clones (SanDI/RsrII) were ligated together at the RsrIIrestriction site. Following transformation into Top10 cells,transformants were screened for the presence of the cassette usingprimers ZeinAmy1199F and Mubi-5 (SY1709: 146, 150). Three positivetransformants were selected and checked via a BamHI diagnostic digest.These clones were sequenced (SY1709:162; both cloning junctions as wellas entire cAmy (SEQ ID NO:52) coding sequence) and it was determinedthat there was a point mutation within the prGZein sequence. Thismutation is a single base pair change-T to C-at nucleotide 340. Thepromoter within the original 15460ZeinAmy backbone was sequenced againto check for the mutation and it was not present in the backbone source.However, it was present when the positive clones were sequenced a secondtime (SY1709:182,189). This mutation may have occurred during bacterialreplication.

The exemplary vector designated “15718,” is a binary vector containingan alpha amylase, the exemplary SEQ ID NO:76, minus its native leader.The native leader was replaced with the Gamma Zein signal sequence fortargeting to the apoplast. The Gamma Zein-SEQ ID NO:76 fusion (cAmy (SEQID NO:76)-02) is expressed using the Gamma Zein promoter. The binaryvector also contains a Ubi-PMI-Nos cassette for mannose selection.

A fragment of cAmy (SEQ ID NO:76)-01, containing amino acids 87-508(plus stop codon), was PCR amplified from plasmid 15650. The PCRamplification introduced an AfeI site at the 5′ end and a BglII site atthe 3′ end of the fragment. The addition of the AfeI site changed aglycine amino acid to alanine. The PCR product was digested with AfeIand BglII and cloned into the 15460AmyZeinVN backbone cut with AfeI andBglII, creating cAmy (SEQ ID NO:76) fused to the gamma zein signalsequence. This intermediate vector was then digested with SanDI andRsrII sequentially. This fragment was gel purified and then ligated into15468 binary vector cut with RsrII and CIP treated. The positive cloneswere PCR size screened and then one of the positive clones was sent forsequence analysis of the entire insert. Sequence was confirmed.

Enzymatic Activity Analysis

Twenty seeds from selected events were randomly selected and pooled. Theseeds were then ground and assayed using the Megazyme CERALPHA HR™ assayfor alpha-amylases or Megazyme's glucoamylase assay for glucoamylases.

Activity Calculations

One CERALPHA™ Unit of α-amylase activity is defined as the amount ofenzyme, in the presence of excess thermostable alpha-glucosidase,required to release one micromole of p-nitrophenol in one minute underthe defined assay conditions. Our assay condition is at pH 5.5 (100 mMNaOAc, pH 5.5 buffer with 1 mg/ml BSA), 60° C. Note that the activitycalculated is a CERALPHA™ unit.

According to Megazyme, 1 mM PNP in 1% tri-sodium phosphate givesabsorbance 18.1 at 400 nm. So 1M of PNP in tri-sodium phosphate givesabsorbance 18,100 at 400 nm.

ΔA₄₀₀/18,100=[PNP] (mol/l or M) in the final plate.

[PNP] in plate (mol/l×8×100×10⁻⁶1×1×10⁶ (umol/mol)=PNP (μmol/rxn)

PNP (μmol/rxn)/(0.05 ml×20 min)=units/ml of diluted alpha-Amylase

units/ml in diluted enzyme×dilution=Ceralpha Units/ml in alpha-Amylasesample

or

ΔA₄₀₀×0.0442×dilution of enzyme=Ceralpha U/ml in alpha-amylase sample.

This SOP is adapted from a CERALPHA HR™ assay method SOP and a Megazymeα-amylase assay procedure (CERALPHA™ method) using amylase HR™ Reagent,ICC Standard No. 303.

FIG. 27 illustrates the theoretical basis of the CERALPHA™ alpha-amylaseassay procedure; from Megazyme's alpha-amylase assay procedure(CERALPHA™ Method) using amylase HR™ reagent: the figure illustrates theoverall reaction scheme: that after alpha-amylase cleaves a bond withinthe blocked p-nitrophenyl malto-saccharide substrate, the non-blockedreaction product containing the p-nitrophenyl constituent is instantlycleaved to glucose and free p-nitrophenyl by the excess quantities ofthermostable alpha-glucosidase, which are integral parts of thesubstrate mixture, and free p-nitrophenyl is released. The reaction isterminated and the phenolate color is developed on addition oftri-sodium phosphate, pH at about 11.0.

Glucoamylase Extraction from Corn Flour and Activity Assay for StarchHydrolysis: Glucoamylase Extraction and Assay from Single Corn Seed

To describe the procedure for glucoamylase extraction and assay fromcorn flour:

Preparation of Extraction Buffer:

25 mM Sodium borate, 0.01% Tween 20, pH 10 buffer

Procedure:

Pooled Seed Grinding:

Pool 20 seeds. Add the seeds to a Kelco grinding vessel. Place a steelball in each vessel on top of the seed. Place a rubber ring around thevessel and place the cap on top. Grind in the Kelco for 45 seconds.Using a scupula transfer the flour from the vessel to a weigh boat andthen pour into a 15 mL conical tube. Wash the vessel, ring, and steelball with soap and water.

Extraction

1. Add 3 ml of extraction buffer to the samples.

2. Seal the tubes with caps using a hammer.

3. Shake vigorously until the flour is suspended in the buffer.

4. Rotate at room temperature on a Rugged rotor at 70% for 5 minutes.

5. Incubate at 45° C. in a water bath for 30 minutes

6. Rotate the samples for another 5 minutes at room temperature afterincubation.

7. Centrifuge the samples at 3000 rpm for 5 minutes.

GA Assay (Reagent Information is after the Assay Procedure)

-   -   1. Dilute samples in the dilution buffer to get an assay in the        range of 0.5 to 1.5 OD₄₀₀.    -   2. Turn on the PCR machine. Set the program to incubate at        40° C. for 10 min and then ramp down to 4° C.    -   3. On ice, add 50 μl of each diluted Glucoamylase sample to a        PCR plate    -   4. Add 150 μl of Stop solution to each well of a microtiter        plate. There should be two wells of Stop solution for each        sample assayed.    -   5. Mix 50 μl of substrate reagent with the sample. Pipette 2        times. Prepare substrate as indicated in the instructions from        Megazyme (10 ml water/bottle).    -   6. Immediately, remove 20 μl of the mixture and mix with 150 μl        of Stop solution. Pipette 3 times with swirling. This is the        0-time point.    -   7. Place plate in the incubator and press Start. Make sure the        samples are in the machine as it ramps up. Incubate the PCR        plate for 10 min at 40° C.    -   8. Allow the machine to ramp down to 4° C. while the samples are        in the machine.    -   9. Wait 2 minutes.    -   10. Remove 20 μl of product and mix with 150 μl of Stop in a        microtiter plate. This is the 10 min. time point.    -   11. Let the plate sit for 15 min.    -   12. Read OD₄₀₀ in the plate reader. Set Pathcheck to ON (this        will normalize the sample absorbance reading as pathlength 1        cm).    -   13. Calculate ΔA₄₀₀, the change in A400 between the 0 and 10 min        point.        Preparation of Stock Solutions:

Substrate (Megazyme catalog #: R-AMGR3) p-nitrophenyl-alpha-maltoside (4mM) Thermostable alpha-glucosidase (5 U/ml)

-   -   Dissolve the entire contents of one vial in 10.0 ml of MilliQ        water. Divide into aliquots and store frozen. Store on ice        during use.

Stop solution: 2% trizma base solution 2.0 g of trizma base (SigmaT-1503) MilliQ water added to final volume of 100 ml

Dilution Buffer: 200 mM Sodium Acetate Buffer (pH 4.5)

Activity Calculations

One Unit of alpha-glucoamylase activity is defined as the amount ofenzyme, in the presence of excess thermostable alpha-glucosidase,required to release one micromole of p-nitrophenol in one minute underthe defined assay conditions.

According to Megazyme, 1 mM PNP in 2% trizma base gives absorbance of18.1 at 400 nm.

$\begin{matrix}{{{GA}\mspace{14mu}{activity}\mspace{14mu}{in}\mspace{14mu}{samples}\mspace{14mu}\left( {U\text{/}{ml}} \right)} = {\left( {A\;{400/10}} \right) \times \left( {170/10} \right) \times \left( {1/18.1} \right) \times {Dilution}}} \\{= {A\; 400 \times 0.0939 \times {Dilution}}}\end{matrix}$

This SOP is adapted from Megazyme's assay of amyloglucosidase usingp-nitrophenyl-alpha-maltoside plus thermostable alpha-glucosidase

Raw Starch Fermentation: carried out as described above.

Results

TAQMAN™ Assay and Enzymatic Activity Assay

Copy numbers of the transgenes were determined by primary and secondaryTAQMAN™ assays. Primers specific for the selectable marker gene used inall the maize transformation vectors described above, pmi, were used inthe primary TAQMAN™ assays. Primers specific for both pmi gene and thebacterial selectable marker gene, spec, were used in secondary TAQMAN™assays.

Expression of alpha-amylases or glucoamylases in mature dried-down T1transgenic seeds of selected constructs were also analyzed by enzymaticactivity assays. Results of gene copy numbers in events generated fromconstruct 15749 and expression of the exemplary alpha-amylase SEQ IDNO:52 in these events were summarized in Table 2.2.

TABLE 2.2 Transgene copy numbers and alpha-amylase activity in eventsgenerated from construct 15749 alpha-amylase Activity in Plant NumberPooled Seed (U/g) 1 25.6 2 23.5 3 21.3 4 21.2 5 20.2 6 18.4 7 15.0 814.3 9 14.0 10 12.7 11 12.2 12 11.1 13 10.2 14 9.9 15 9.6 16 7.6 17 6.918 6.8 19 6.4 20 6.4 21 6.0 22 5.4 23 5.3 24 5.3 25 4.7 26 4.7 27 4.5 284.2 29 4.0 30 3.9 31 3.7 32 3.6 34 3.1 35 3.0 36 2.8 37 2.5 38 2.4 392.0 40 1.5 41 0.4 42 0.4 43 0.3 44 0.2 45 0.1

Results of gene copy numbers in events generated from construct 15751and expression of glucoamylase SEQ ID NO:48 in these events aresummarized in Table 2.3:

TABLE 2.3 Transgene copy numbers and glucoamylase activity in eventsgenerated from construct 15751 Gluco-amylase Activity in Pooled PlantSeed (U/g) 1 4.86 2 4.48 3 4.47 4 4.22 5 4.20 6 4.08 7 4.05 8 3.94 93.88 10 3.84 11 3.82 12 3.68 13 3.65 14 3.57 15 3.50 16 3.47 17 3.43 183.33 19 2.91 20 1.13 21 0.73

Table 2.4 summarizes results of gene copy numbers in events generatedfrom construct 15756 and expression of glucoamylase SEQ ID NO:26 inthese events. Seeds from multiple events of constructs 15761 and 15718expressing alpha-amylases SEQ ID NO:66 and SEQ ID NO:76, respectively,were pooled and ground to make composite samples. The two compositesamples were assayed for □-amylase activities. Similarly, seeds frommultiple events of constructs 15742 and 15750 expressing glucoamylasesSEQ ID NO:28 and SEQ ID NO:18, respectively, were also pooled and groundto make composite samples. The two composite samples were assayed forglucoamylase activities. The results were summarized in Table 2.5.

TABLE 2.4 Transgene copy numbers and glucoamylase activity in eventsgenerated from construct 15756 Gluco-amylase Activity in Pooled SeedPlant number (U/g) 1 4.41 2 4.31 3 4.18 4 4.15 5 3.95 6 3.94 7 3.88 83.83 9 3.78 10 3.68 11 3.67 12 3.64 13 3.60 14 3.58 15 3.45 16 3.27 173.18 18 3.16 19 3.14 20 3.10 21 3.07 22 3.00 23 2.75

TABLE 2.5 alpha-amylase or glucoamylase activity in seeds generated fromconstructs 15718, 15761, 15742, and 15750 Pooled Seed Activity ConstructVariety Enzyme Expressed Enzyme Type (U/g) 15718 JHAX707 SEQ ID NO: 76alpha-amylase 0.033 15761 JHAX708 SEQ ID NO: 66 alpha-amylase 0.08515742 JHAX709 SEQ ID NO: 28 Glucoamylase 3.399 15750 JHAX710 SEQ ID NO:18 Glucoamylase 3.55Raw Starch Fermentation Using Maize Expressed Enzymes

Seeds from multiple events of selected constructs expressingalpha-amylases or glucoamylases were pooled and ground to make compositesamples for raw starch fermentations. In all experiments, corn flourcontaining alpha-amylases was used at the inclusion rate of 20% (w/w)and corn flour containing glucoamylases at the inclusion rate of 50%(w/w). Yellow dent II commodity corn flour made up the rest of the 30%(w/w) of the corn flour. Raw starch fermentations were carried out byfollowing the standard SOP described above (Section I). The ability ofthese maize expressed alpha-amylase and glucoamylase combinations toproduce ethanol in raw starch fermentation was summarized in Table 2.6.

TABLE 2.6 Ethanol yield after 72 hours of raw starch fermentation using“lead” exemplary enzymes expressed in maize without codon optimization*Inclusion Inclusion Rate Inclusion Rate (alpha- Rate (Commodity) EthanolConstruct Construct amylase) (Glucoamylase) (% Yield (alpha-amylase)(Glucoamylase) (% w/w) (% w/w) w/w) (% v/v) 15749 (SEQ ID 15756 (SEQ ID20 50 30 6.96 NO: 52) NO: 26) 15749 (SEQ ID 15742 (SEQ ID 20 50 30 8.90NO: 52) NO: 28) 15761 (SEQ ID 15751 (SEQ ID 20 50 30 7.30 NO: 66) NO:48) 15718 (SEQ ID 15750 (SEQ ID 20 50 30 8.33 NO: 76) NO: 18) N/A N/A 00 100 2.25

Example 28: Plant Expression of Exemplary Enzymes Using Synthetic MaizeCodon Optimized Genes and Raw Starch Fermentation Using Plant Material

Synthetic genes with codons optimized for maize expression weregenerated for 2 alpha-amylases, SEQ ID NO:52 (encoded, e.g., by SEQ IDNO:51) and SEQ ID NO:4 (encoded, e.g., by SEQ ID NO:3), and 2glucoamylases, SEQ ID NO:48 (encoded, e.g., by SEQ ID NO:47) and SEQ IDNO:26 (encoded, e.g., by SEQ ID NO:25). The table below correlates thewild-type SEQ ID NO: with the SEQ ID NO: assigned to the codon-optimizedsequence. The enzymes chosen for codon optimization were selected basedon giving the highest ethanol yield in raw starch fermentation. Theobjectives of this study were to generate commercial events for productdevelopment and to generate enough material to evaluate the raw starchfermentation performance of the maize expressed enzymes.

Codon-optimized Enzyme Wild-type SEQ ID NO: SEQ ID NO: Amylase 51 79Amylase 3 80 Glucoamylase 47 81 Glucoamylase 25 82

Experimental Methods

Synthetic Genes

The synthetic genes were made by GENEART™. The sequences were codonoptimized using GENEART™'s proprietary GENEOPTIMIZER™ technology.Sequences of the synthetic genes are listed below. The codon usage wasadapted to the codon bias of Zea mays genes.

Vector Construction

Table 3.1 summarized the abbreviations used for the DNA elements in themaps shown below.

TABLE 3.1 Abbreviations used in the commercial construct maps NameFunction Description cAmy(SEQ ID CDS Apoplast targeted maize codonoptimized synthetic NO: 79)Apo-01 alpha-amylase gene encoding enzyme SEQID NO: 52 cAmy(SEQ ID CDS ER targeted synthetic maize codon-optimizedalpha- NO: 79)ER-01 amylase gene with Gamma Zein signal sequence andKDEL sequence encoding enzyme SEQ ID NO: 52 cAmy(SEQ ID CDS Apoplasttargeted synthetic maize codon optimized NO: 80)Apo-01 alpha-amylasegene encoding enzyme SEQ ID NO: 4 cAmy(SEQ ID CDS ER targeted syntheticmaize codon-optimized alpha- NO: 80)ER-01 amylase gene with Gamma Zeinsignal sequence and KDEL sequence encoding enzyme SEQ ID NO: 4 cGAmy(SEQID CDS Apoplast targeted synthetic maize codon optimized NO: 82)Apo-01glucoamylase gene encoding enzyme SEQ ID NO: 26 cGAmy(SEQ ID CDS ERtargeted synthetic maize codon optimized NO: 82)ER-01 glucoamylase genewith gamma zein signal sequence and KDEL sequence encoding enzyme SEQ IDNO: 26 cGAmy(SEQ ID CDS Apoplast targeted synthetic maize codonoptimized NO: 81)Apo-01 gluco-amylase gene encoding enzyme SEQ ID NO: 48cGAmy(SEQ ID CDS ER targeted (with addition of KDEL ER-retention NO:81)ER-01 signal) synthetic maize codon optimized glucoamylase geneencoding enzyme SEQ ID NO: 48

The exemplary vector designated “15740,” is a binary vector for maizetransformation harboring seed specific promoter prGTL-03 driving theapoplast targeted synthetic maize optimized version of the exemplaryalpha-amylase SEQ ID NO:51, the optimized version is the exemplary SEQID NO:79 or, cAmy (SEQ ID NO:79) Apo-01. This binary also contains aUbi-PMI-Nos cassette for selection.

Cloning Vector: Construct alias15460ZeinAmyVN was digested with SacI andRsrII to remove iPEPC9-01 and t35s-08. Likewise, construct 15460 wasdigested with SacI and RsrII to remove tNOS-03-01. Terminatort35s-08/iPEPC9-01 was ligated into 15460 using these sites (SY1709:6).The ligation reaction was transformed into DH5-alpha cells;transformants were screened with colony PCR. Four positive clones weresequenced, and from these sequences both cloning junctions andrestriction enzyme sites were determined to be correct (SY1709:15-16).To insert promoter prGTL-03, 15460 containing t35s08/iPEPC9-01 wasdigested with BamHI and HindIII and gel purified (SY1709:14). Construct11267 containing prGTL-03 was also digested with BamHI and HindIII andthe promoter was gel purified (SY1710:3-4). It was then ligated into the15460 backbone using these sites and transformed into competentDH5-alpha cells. Ten transformants were screened using HindIII and BamHIto determine if prGTL-03 was present in vector; all ten had correctbanding pattern (SY1709:25). Two clones were sequenced to confirmcloning junctions and restriction enzyme sites, and both were correctwithout any sequence differences (SY1709:34-35). Glycerol stocks wereprepared from clone #1 and stored at −80 degrees Celsius. Cloning vectorwas digested with BamHI/SacI and CIP treated. The synthetic maize codonoptimized version of alpha amylase SEQ ID NO:51, known as SEQ ID NO:79(also known as cAmy (SEQ ID NO:79)) was digested with BamHI and SacI andligated into the BamHI/SacI site of prGTL-03 cloning vector to createalias GTL+SYN(SEQ ID NO:79) (Sy1773:48). Binary Vector: An AscI/BamHIfragment from vector 15468 was ligated into the AscI/BamHI site ofbinary vector 12678 to create alias “12678 RsrII” (SY1533:189). Thisclone was then digested with RsrII and CIP treated. Cloning vector“GTL+SYN(SEQ ID NO:79)” was digested with SanDI/RsrII and ligated intothe RsrII site to create B-prGTL:(SEQ ID NO:79):t35S:PMI (SY1773:56).Positive clones were identified by PCR and confirmed by DNA sequencing.

The exemplary vector designated “15741,” is a binary vector for maizetransformation harboring seed specific promoter prGTL-03 driving theapoplast targeted synthetic maize optimized version of the exemplaryglucoamylase SEQ ID NO:25, the optimized version is the exemplary SEQ IDNO:82 or cGAmy (SEQ ID NO:82) Apo-01. This binary also contains aUbi-PMI-Nos cassette for selection.

Cloning Vector: Construct alias15460ZeinAmyVN was digested with SacI andRsrII to remove iPEPC9-01 and t35s-08. Likewise, construct 15460 wasdigested with SacI and RsrII to remove tNOS-03-01. Terminatort35s-08/iPEPC9-01 was ligated into 15460 using these sites (SY1709:6).The ligation reaction was transformed into DH5-alpha cells;transformants were screened with colony PCR. Four positive clones weresequenced, and from these sequences both cloning junctions andrestriction enzyme sites were determined to be correct (SY1709:15-16).To insert promoter prGTL-03, 15460 containing t35s08/iPEPC9-01 wasdigested with BamHI and HindIII and gel purified (SY1709:14). Construct11267 containing prGTL-03 was also digested with BamHI and HindIII andthe promoter was gel purified (SY1710:3-4). It was then ligated into the15460 backbone using these sites and transformed into competentDH5-alpha cells. Ten transformants were screened using HindIII and BamHIto determine if prGTL-03 was present in vector; all ten had correctbanding pattern (SY1709:25). Two clones were sequenced to confirmcloning junctions and restriction enzyme sites, and both were correctwithout any sequence differences (SY1709:34-35). Glycerol stocks wereprepared from clone #1 and stored at −80 degrees Celsius. Cloning vectorwas digested with BamHI/SacI and CIP treated. The synthetic maize codonoptimized version of glucoamylase SEQ ID NO:25, known as SEQ ID NO:82(also known as cAmy (SEQ ID NO:82 or SYN(SEQ ID NO:82) was digested withBamHI and SacI and ligated into the BamHI/SacI site of prGTL-03 cloningvector to create alias GTL+SYN(SEQ ID NO:82) (SY1773:48). Binary Vector:An AscI/BamHI fragment from vector 15468 was ligated into the AscI/BamHIsite of binary vector 12678 to create alias “12678 RsrII” (SY1533:189).This clone was then digested with RsrII and CIP treated. Cloning vector“GTL+SYN(SEQ ID NO:82)” was digested with SanDI/RsrII and ligated intothe RsrII site to create B-prGTL:(SEQ ID NO:82):t35S:PMI (SY1773:66).Positive clones were identified by PCR and confirmed by DNA sequencing.

The exemplary vector designated “15742” is a binary vector harboring(comprising) the seed specific promoter prGTL-03 driving the apoplasttargeted synthetic maize optimized version of the exemplary glucoamylaseSEQ ID NO:47, the optimized version is the exemplary SEQ ID NO:81. Thisbinary also contains a Ubi:PMI:NOS cassette for selection.

Cloning Vector: Construct alias15460ZeinAmyVN was digested with SacI andRsrII to remove iPEPC9-01 and t35s-08. Likewise, construct 15460 wasdigested with SacI and RsrII to remove tNOS-03-01. Terminatort35s-08/iPEPC9-01 was ligated into 15460 using these sites (SY1709:6).The ligation reaction was transformed into DH5-alpha cells;transformants were screened with colony PCR. Four positive clones weresequenced, and from these sequences both cloning junctions andrestriction enzyme sites were determined to be correct (SY1709:15-16).To insert promoter prGTL-03, 15460 containing t35s08/iPEPC9-01 wasdigested with BamHI and HindIII and gel purified (SY1709:14). Construct11267 containing prGTL-03 was also digested with BamHI and HindIII andthe promoter was gel purified (SY1710:3-4). It was then ligated into the15460 backbone using these sites and transformed into competentDH5-alpha cells. Ten transformants were screened using HindIII and BamHIto determine if prGTL-03 was present in vector; all ten had correctbanding pattern (SY1709:25). Two clones were sequenced to confirmcloning junctions and restriction enzyme sites, and both were correctwithout any sequence differences (SY1709:34-35). Glycerol stocks wereprepared from clone #1 and stored at −80 degrees Celsius. Cloning vectorwas digested with BamHI/SacI and CIP treated. The synthetic maize codonoptimized version of glucoamylase SEQ ID NO:47, known as SEQ ID NO:81(also known as cAmy (SEQ ID NO:81) was digested with BamHI and SacI andligated into the BamHI/SacI site of prGTL-03 cloning vector to createalias GTL+SYN(SEQ ID NO:81)) (SY1773:48). Binary Vector: An AscI/BamHIfragment from vector 15468 was ligated into the AscI/BamHI site ofbinary vector 12678 to create alias “12678 RsrII” (SY1533:189). Thisclone was then digested with RsrII and CIP treated. Cloning vector“GTL+SYN(SEQ ID NO:81)” was digested with SanDI/RsrII and ligated intothe RsrII site to create B-prGTL:(SEQ ID NO:81):t35S:PMI (SY1773:56).Positive clones were identified by PCR and confirmed by DNA sequencing(SY1773:58).

The exemplary vector designated “15743,” is a binary vector harboring(comprising) the seed specific promoter prGTL-03 driving the apoplasttargeted synthetic maize optimized version of the exemplaryalpha-amylase SEQ ID NO:3, the optimized version is SEQ ID NO:80. Thisbinary also contains a Ubi:PMI:NOS cassette for selection.

Cloning Vector: Construct alias15460ZeinAmyVN was digested with SacI andRsrII to remove iPEPC9-01 and t35s-08. Likewise, construct 15460 wasdigested with SacI and RsrII to remove tNOS-03-01. Terminatort35s-08/iPEPC9-01 was ligated into 15460 using these sites (SY1709:6).The ligation reaction was transformed into DH5-alpha cells;transformants were screened with colony PCR. Four positive clones weresequenced, and from these sequences both cloning junctions andrestriction enzyme sites were determined to be correct (SY1709:15-16).To insert promoter prGTL-03, 15460 containing t35s08/iPEPC9-01 wasdigested with BamHI and HindIII and gel purified (SY1709:14). Construct11267 containing prGTL-03 was also digested with BamHI and HindIII andthe promoter was gel purified (SY1710:3-4). It was then ligated into the15460 backbone using these sites and transformed into competentDH5-alpha cells. Ten transformants were screened using HindIII and BamHIto determine if prGTL-03 was present in vector; all ten had correctbanding pattern (SY1709:25). Two clones were sequenced to confirmcloning junctions and restriction enzyme sites, and both were correctwithout any sequence differences (SY1709:34-35). Glycerol stocks wereprepared from clone #1 and stored at −80 degrees Celsius. Cloning vectorwas digested with BamHI/SacI and CIP treated. The synthetic maize codonoptimized version of alpha-amylase SEQ ID NO:3, known as SEQ ID NO:80(also known as cAmy (SEQ ID NO:80) was digested with BamHI and SacI andligated into the BamHI/SacI site of prGTL-03 cloning vector to createalias GTL+SYN(SEQ ID NO:80) (SY1773:48). Binary Vector: An AscI/BamHIfragment from vector 15468 was ligated into the AscI/BamHI site ofbinary vector 12678 to create alias “12678 RsrII” (SY1533:189). Thisclone was then digested with RsrII and CIP treated. Cloning vector“GTL+SYN(SEQ ID NO:80)” was digested with SanDI/RsrII and ligated intothe RsrII site to create B-prGTL:(SEQ ID NO:80):t35S:PMI (SY1773:56).Positive clones were identified by PCR and confirmed by DNA sequencing.

The exemplary vector designated “15862,” is a binary vector for maizetransformation harboring (comprising) the seed specific promoterprGTL-03 driving the synthetic maize optimized version of the exemplaryglucoamylase SEQ ID NO:47, the optimized version is the exemplary SEQ IDNO:81 or cGAmy (SEQ ID NO:81)ER-01 that is targeted to the ER by theKDEL ER-retention signal. This binary also contains a Ubi-PMI-Noscassette for selection.

The maize codon optimized version of SEQ ID NO:47, known as SYN(SEQ IDNO:81) (cAMY(SEQ ID NO:81)-03) was PCR replicated and TOPO cloned withprimers such that an ER-retention signal KDEL was added to the 3-primeend (SY1773:53). The presence of the KDEL was confirmed by DNAsequencing (SY1773:54 & 60). DNA sequence data confirmed nodiscrepancies (SY1773:63). Clone #1 was digested with BamHI/BglII,purified by agarose gel and ligated into a cloning vector harboring theprGTL-03 promoter and t35S-08 to create alias “GTL+SYN(SEQ ID NO:81)kdel v2” (SY1773:66). A gene cassette prGTL-03:cAmy (SEQ ID NO:81)kdel:t35s-08 was ligated as a SanDI/RsrII fragment into the RsrII siteof a modified version of binary vector 12678 to create B-prGTL-(SEQ IDNO:81) KDEL:PMI (SY1773:83). Integrity of the binary vector wasconfirmed by PCR and DNA sequencing (SY1773:88).

The exemplary vector designated “15880,” is a binary vector containing(comprising) the maize codon-optimized version of the exemplary alphaamylase SEQ ID NO:51, the codon-optimized version is the exemplary SEQID NO:79, was expressed using the rice glutelin promoter (prGTL-03). Thealpha amylase also contains a Gamma Zein signal sequence and KDELsequence for ER retention. This vector contains an Ubi-PMI-Nos cassettefor mannose selection.

The maize codon optimized alpha amylase SEQ ID NO:51, known as SEQ IDNO:79 or cAmy (SEQ ID NO:79) was PCR replicated with primers such thatan ER-retention signal (KDEL) was added to the 3-prime end (SY1777:180).The PCR product was then gel purified, TOPO cloned, and transformed intoTOP10 competent cells. Presence of the KDEL without any discrepancieswas confirmed via sequencing (SY1773:73). TOPO clones were digested withBamHI and BglII to obtain the modified amylase gene which was then gelpurified. The gel purified product was ligated into 15460 containingprGTL-03 (rice glutelin promoter) and t35s-08 to create alias “GTL+syn(SEQ ID NO:79) KDEL v2” (SY1773:80; SY1777:180). Followingtransformation into DH5a competent cells, clones were screened forinsert orientation via PCR. In addition, the entire CDS as well ascloning junctions were sequenced prior to construction of binary vector(SY1777:189, 191). The gene cassette prGTL:cAmy (SEQ ID NO:79):t35s08was digested with SanDI/RsrII and ligated into a modified version ofconstruct 12678 at the RsrII site (SY1818:12). This construct wasmodified by ligating an AscI/BamHI fragment from vector 15468 into theAscI/BamHI site of binary vector 12678 to create alias “12678 RsrII”(SY1533:189). The ligation reaction was transformed into TOP10 competentcells, and transformants were subsequently screened via colony PCR. Twoof the screened transformants were positive for the gene cassette andwere digested with BamHI to confirm its presence. Following the digestconfirmation, the entire cassette from clone #5 was sequenced(SY1818:34). Data confirmed that the entire cassette, including alljunctions, is present without any sequence discrepancies (SY1818:37-38).

The exemplary vector designated “15884,” is a binary vector containing(comprising) the maize codon-optimized version of the exemplary SEQ IDNO:25, the maize codon-optimized version is the exemplary SEQ ID NO:82,was expressed using the rice glutelin promoter (prGTL-03). Theglucoamylase also contains a Gamma Zein signal sequence and ER retentionsignal (KDEL). This vector contains an Ubi-PMI-Nos cassette for mannoseselection.

The maize codon optimized version glucoamylase SEQ ID NO:25, known asSEQ ID NO:82 or cAmy (SEQ ID NO:82) was PCR replicated with primers suchthat an ER-retention signal (KDEL) was added to the 3-prime end(SY1777:184). The PCR product was then gel purified, TOPO cloned, andtransformed into TOP10 competent cells. Presence of the KDEL without anydiscrepancies was confirmed via sequencing (SY1777:190). TOPO cloneswere digested with BamHI and BglII to obtain the modified amylase genewhich was then gel purified. The gel purified product was ligated into15460 containing prGTL-03 (rice glutelin promoter) and t35s-08 to createalias “GTL+syn (SEQ ID NO:82) KDEL v2” (SY1818; 1-3). Followingtransformation into Top10 competent cells, clones were screened forinsert orientation via PCR. In addition, the entire CDS as well ascloning junctions were sequenced prior to construction of binary vector(SY1818:29-30). It was determined from sequencing that there is a singlebase pair change within the coding sequence of cAmy (SEQ ID NO:82) atbase pair 156. It is a T to C change that does not change the amino acidsequence of the protein (SY1818: 39-40). The gene cassette prGTL: syn(SEQ ID NO:82) KDEL: t35s08 was digested with SanDI/RsrII and ligatedinto a modified version of construct 12678 at the RsrII site(SY1818:31). This construct was modified by ligating an AscI/BamHIfragment from vector 15468 into the AscI/BamHI site of binary vector12678 to create alias “12678 RsrII” (SY1533:189). The ligation reactionwas transformed into TOP10 competent cells, and transformants weresubsequently screened using restriction enzyme digests. Two of thetransformants were digested with various combinations of restrictionenzymes including NotI, SacI, EcoRV, and PstI to confirm the presence ofthe gene cassette (SY1818:45). Following the digest confirmation, theentire cassette from clone #7 was sequenced (SY1818:46-47). Dataconfirmed that the entire cassette, including all junctions, is presentand only contains the single base pair change described earlier(SY1818:50).

The exemplary vector designated “15890,” is a binary vector for maizetransformation containing the maize codon-optimized version of theexemplary alpha-amylase SEQ ID NO:3, the maize codon-optimized versionis the exemplary SEQ ID NO:80 or cAmy (SEQ ID NO:80) was expressed usingthe rice glutelin promoter (prGTL-03). The glucoamylase also contains aGamma Zein signal sequence and ER retention signal (KDEL). This vectorcontains an Ubi-PMI-Nos cassette for mannose selection.

The maize codon optimized version of the exemplary alpha-amylase SEQ IDNO:3, known as cAmy (SEQ ID NO:80) was PCR replicated with primers suchthat an ER-retention signal (KDEL) was added to the 3-prime end(SY1777:184). The PCR product was then gel purified, TOPO cloned, andtransformed into TOP10 competent cells. Presence of the KDEL without anydiscrepancies was confirmed via sequencing (SY1777:190). TOPO cloneswere digested with BamHI and BglII to obtain the modified amylase genewhich was then gel purified. The gel purified product was ligated into15460 containing prGTL-03 (rice glutelin promoter) and t35s-08 to createalias “GTL+syn (SEQ ID NO:80) KDEL v2” (SY1818; 1-3). Followingtransformation into TOP10 competent cells, clones were screened forinsert orientation via PCR. In addition, the entire CDS as well ascloning junctions were sequenced prior to construction of binary vector(SY1818:51). The gene cassette prGTL: syn (SEQ ID NO:80) KDEL: t35s08was digested with SanDI/RsrII and ligated into a modified version ofconstruct 12678 at the RsrII site (SY1818:52). This construct wasmodified by ligating an AscI/BamHI fragment from vector 15468 into theAscI/BamHI site of binary vector 12678 to create alias “12678 RsrII”(SY1533:189). The ligation reaction was transformed into TOP10 competentcells, and transformants were subsequently screened using PCR andrestriction enzyme digests. Two of the transformants that had positivePCR results were digested with various combinations of restrictionenzymes including NcoI, KpnI, EcoRV, XbaI, and BglII to confirm thepresence of the gene cassette (SY1818:65-66). Following the digestconfirmation, the entire cassette from clone #2 was sequenced(SY1818:67). Data confirmed that the entire cassette, including alljunctions, is present without any sequence differences (SY1818:69-70).

The exemplary vector designated “15889,” is a binary vector for maizetransformation containing a molecular stack of the codon optimizedversion of the exemplary glucoamylase SEQ ID NO:25, the codon optimizedversion is the exemplary SEQ ID NO:82 or cGAmy (SEQ ID NO:82) wasexpressed by two different promoters. In the first cassette, cAmy (SEQID NO:82) is driven by the rice glutelin promoter and has a Gamma Zeinsignal sequence and KDEL signal for retention in the ER. In the secondcassette, the gluco-amylase is driven by the alpha trypsin inhibitorpromoter and is targeted to the apoplast with the Gamma Zein signalsequence. This construct also contains a Ubi-PMI-Nos cassette formannose selection.

The rice promoter, prATI-01 was mutagenized through site-directedmutagenesis to remove an internal RsrII site, and was submitted as a newcomponent in construct #15882. Following sequence confirmation of theremoval of this site, the cloning vector “15460+prATI+t35s08” wasdigested with BamHI/SacI, CIP treated, and gel extracted (SY1777:162).The synthetic, codon optimized gluco-amylase Syn (SEQ ID NO:82)(BamHI/SacI) was ligated into the digested backbone and the resultingconstruct is known as “15460prATIm.syn (SEQ ID NO:82)” (SY1777:170).Transformants were screened for the presence of the gene, using bothcolony PCR and a diagnostic NcoI/BglII digest. Three positive cloneswere sequenced; cloning junctions were confirmed for the three clones(SY1777:177-179). Following sequence confirmation, “15460prATIm.syn (SEQID NO:82)” was digested with SanDI/RsrII and the ATI: syn (SEQ IDNO:82):t35s08 cassette was gel purified (SY1773:52). Binary vector#15884 was linearized with RsrII, CIP treated, and gel extracted.Following purification, the ATI: syn (SEQ ID NO:82):t35s08 cassette wasligated into the binary backbone and transformed into TOP10 competentcells (SY1818:53). Ten transformants were selected for screening, viaPCR, one of which was confirmed to have the cassette containing the ATIpromoter (SY1818:56). The positive clone was then screened withdiagnostic restriction enzyme digests using the following enzymes:EcoRI, BglII, NcoI, and HindIII (SY1818:60, 66). Clone #9 was determinedto be correct by analyzing the banding pattern from the various digests.In addition, the cloning junctions of clone #9 were sequenced and alsoconfirmed to be correct (SY1818:65).

The exemplary vector designated “15934,” is a binary vector for planttransformation containing a molecular stack of the codon optimizedversion of the exemplary glucoamylase SEQ ID NO:47, the codon optimizedversion is the exemplary SEQ ID NO:81 or cGAmy (SEQ ID NO:81) wasexpressed by two different promoters. In the first cassette, GAmy (SEQID NO:81) is driven by the rice glutelin promoter and has a KDEL signalfor retention in the ER (component is cGAmy (SEQ ID NO:81)ER-01). In thesecond cassette, the GAmy (SEQ ID NO:81) is driven by the alpha trypsininhibitor promoter and is targeted to the apoplast (component is cGAmy(SEQ ID NO:81) Apo-01). This construct also contains an Ubi-PMI-Noscassette for mannose selection. NOTE: IT was determined by restrictiondigest and sequencing that one of the left border insertions wasmissing. The project elected to proceed with the vector.

The cloning vector “15460+prATI+t35s08” was digested with BamHI/SacI,CIP treated, and gel extracted (SY1777:162). The synthetic, codonoptimized gluco-amylase cGAmy (SEQ ID NO:81) Apo-01 (BamHI/SacI) wasligated into the digested backbone and the resulting construct is knownas “15460prATIm.syn (SEQ ID NO:81)” (SY1777:170). Transformants werescreened for the presence of the gene, using both colony PCR and adiagnostic NcoI/BglII digest. Three positive clones were sequenced;cloning junctions were confirmed for the three clones (SY1777:177-179).Following sequence confirmation, “15460prATIm.syn (SEQ ID NO:81)” wasdigested with SanDI/RsrII and the ATI: cGAmy (SEQ ID NO:81)Apo-01:t35s08 cassette was gel purified (SY1773:72). Binary vector“B-prGTL: (SEQ ID NO:81) KDEL: PMI” was constructed and labeled asconstruct #15862 (SY17773:92). It was linearized with RsrII, CIPtreated, and gel extracted. Following purification, the ATI: cGAmy (SEQID NO:81) Apo-01:t35s08 cassette was ligated into the binary backboneand transformed into TOP10 competent cells (SY1818:22-23). Transformantswere initially screened with colony PCR using primers which onlyannealed in the ATI promoter. Ten transformants were selected andconfirmed to have the cassette containing the ATI promoter. Clone #8 wasselected for restriction analyses to further confirm the presence of thecassette within the binary vector. Two enzymes which specifically cut inthe ATI promoter as well as the backbone were selected to rule out thepossibility of a double cassette. When clone #8 was digested with AvrIIand NcoI, it was determined that there was only one copy of eachcassette present (SY1818: 93-94).

Enzymatic Activity Analysis

Single seed analysis or pooled seed analysis was used to measurealpha-amylase or glucoamylase activity in transgenic seeds. For singleseed analysis, 12 seeds from each event were randomly selected andground individually. For pooled seed analysis, twenty seeds fromselected events were randomly selected, pooled, and ground. The flourswere then assayed using the Megazyme CERALPHA HR™ assay foralpha-amylases or Megazyme's glucoamylase assay for glucoamylases. Theassay's standard operating procedures (SOPs) are described above,including the “enzymatic activity analysis”, the alpha-amylaseextraction from corn flour and activity assay, the glucoamylaseextraction from corn flour and activity assay, and the raw starchfermentation protocol.

Results

Taqman Assay and Enzymatic Activity Assay

Copy numbers of the transgenes were determined by primary and secondaryTaqman assays. Primers specific for the selectable marker gene used inall the maize transformation vectors described above, pmi, were used inthe primary Taqman assays. Primers specific for pmi gene, the bacterialselectable marker gene, spec, and genes encoding the alpha-amylases orglucoamylases were used in secondary Taqman assays.

Expression of alpha-amylases or glucoamylases in mature dried-down T1transgenic seeds of selected constructs were also analyzed by enzymaticactivity assays. Results of gene copy numbers in selected eventsgenerated from construct 15840 were summarized in Table 3.2. Expressionof the exemplary codon-optimized alpha-amylase SEQ ID NO:79 in theseevents were measured by analysis of enzymatic activity in 12 randomlyselected seeds individually. The average activity of the 12 seeds foreach event is also shown in Table 3.2, below.

Similarly, Table 3.3 shows gene copy numbers of selected eventsgenerated from construct 15841. Expression of the exemplarycodon-optimized glucoamylase SEQ ID NO:82 in these events were alsomeasured by analysis of enzymatic activity in 12 randomly selected seedsindividually and average activity of the 12 seeds for each event issummarized in Table 3.3, below.

Twenty (20) seeds from each selected events generated from constructs15842 and 15843, on the other hand, were pooled for enzymatic assays todetermine expression levels of the codon-optimized glucoamylase SEQ IDNO:81 and the exemplary codon-optimized alpha-amylase SEQ ID NO:80 inthese events, respectively. Results were summarized in Tables 3.4 and3.5, respectively, below. Transgene copy numbers are also shown.

TABLE 3.2 Transgene copy numbers and alpha-amylase activity in selectedevents generated from construct 15840 Plant number Average Activity of12 Seeds (U/g) 1 377.3 2 291.9 3 95.1 4 119.4 5 155.4 6 129.4 7 125.3 8539.4 9 1362.4 10 195.1 11 88.4 12 458.2 13 722.9 14 151.7 15 164.8

TABLE 3.3 Transgene copy numbers and glucoamylase activity in selectedevents generated from construct 15841 Average Activity of 12 PlantNumber Seeds (U/g) 1 2.5 2 3.5 3 2.6 4 2.8 5 2.4 6 3.0 7 2.6 8 2.5 9 2.610 2.8 11 3.0 12 3.0 13 2.9 14 3.4 15 2.3 16 2.5 17 2.6 18 3.3 19 2.4 202.9 21 3.5 22 2.3 23 2.5 24 2.3

TABLE 3.4 Transgene copy numbers and glucoamylase activity in selectedevents generated from construct 15842 Plant Number Pooled Seed Activity(U/g) 1 1.5 2 1.3 3 1.3 4 1.2 5 1.3 6 1.6 7 1.4 8 1.4 9 1.3 10 1.2 111.4 12 1.6 13 1.7 14 1.2 15 1.3 16 1.6 17 1.4 18 1.6 19 1.6 20 1.6

TABLE 3.5 Transgene copy numbers and alpha-amylase activity in selectedevents generated from construct 15843 Plant Number Pooled Seed Activity(U/g) 1 116.14 2 107.07 3 105.02 4 277.65 5 240.90 6 165.25 7 425.17 8151.02 9 139.74 10 245.27 11 197.68 12 179.77 13 368.59 14 350.91 15225.03 16 233.18 17 366.24 18 220.36

TABLE 3.6 Ethanol yield after 72 hours of raw starch fermentation using“lead” exemplary enzymes expressed in maize using maize codon optimizedsynthetic genes Inclusion Inclusion Construct Rate (alpha- InclusionRate Rate (alpha- Construct amylase) (Glucoamylase) (Commidity) Ethanolamylase) (Glucoamylase) (% w/w) (% w/w) (% w/w) Yield (% v/v) 1584015841 (SEQ ID 20 50 30 16.77 (SEQ ID NO: 82) NO: 79) 15840 15842 (SEQ ID20 50 30 17.08 (SEQ ID NO: 81) NO: 79) 15843 15841 (SEQ ID 20 50 30 7.30(SEQ ID NO: 82) NO: 80) 15843 15842 (SEQ ID 20 50 30 10.60 (SEQ ID NO:81) NO: 80) N/A N/A 0 0 100 2.25

Raw Starch Fermentation Using Maize Expressed Enzymes

Seeds from events shown above expressing alpha-amylases or glucoamylaseswere pooled and ground to make composite samples for raw starchfermentations. In all experiments, corn flour containing alpha-amylaseswas used at the inclusion rate of 20% (w/w) and corn flour containingglucoamylases at the inclusion rate of 50% (w/w). Yellow dent IIcommodity corn flour made up the rest of the 30% (w/w) of the cornflour. Raw starch fermentations were carried out by following thestandard SOP described above (Section I). The ability of these maizeexpressed alpha-amylase and glucoamylase combinations using maize codonoptimized synthetic genes to produce ethanol in raw starch fermentationis summarized in Table 3.6, below.

Example 29: Pichia pastoris Expression Constructs

This example described the expression of enzymes in yeast using, e.g.,exemplary Pichia expression systems.

For construction of expression constructs in pPICZalpha and pAO815vectors (both from Invitrogen, Carlsbad, Calif.), Xi-cloning technologywas used. pPICZalpha was digested with EcoRI, then treated withXi-cloning cocktails (according to manufacture protocol by Genlantis, adivision of Gene Therapy Systems, Inc., San Diego, Calif.). The geneswere amplified by PCR reactions with end matching to the vectorsequences. The PCR products were mixed with the vectors and transformedinto E. coli host TOP10 (Invitrogen) and selected under zeocin 25(pPICZalpha) or carbenicillin 100 (pAO815). The final constructs wereverified by sequencing. For transformation into Pichia hosts, theverified plasmid DNA was digested to make linear DNA and transform in toPichia hosts. The transformants were selected under Zeocin (pPICZalpha)or histine deficient plates.

All enzymes were obtained by expression in Pichia pastoris, except forSEQ ID NO:78 (encoded by SEQ ID NO:77), which was expressed inPseudomonas fluorescens (see JBC, 2002, 277(29):26501-26507).

I. Characterization of Glucoamylases and Amylases.

Methods:

6. Determination of Protein Concentration

Lyophilized supernatants of P. pastoris cultures expressingglucoamylases and alpha-amylases were suspended in water at aconcentration of ˜10 mg of powder/ml. After protein contentdetermination by the Bradford protocol, 5 μg of protein sample andstandardized BSA solution were run on a 4-20% Tris-Glycine gradient gel.The gel was scanned on the BioRad GS800 gel scanner following Coomassieblue staining. The Bio-Rad Quantity One software was used for thequantification of the BSA and glucoamylase (or alpha-amylases) bands,and the actual enzyme concentration was then calculated. Proteinconcentration was adjusted accordingly and confirmed by additional SDSPAGE.

7. Determination of Initial Reaction Rates.

Unless mentioned otherwise, assays were performed in triplicate at 37°C. and pH 5.0 in buffer (50 mM NaCH₃CO₂, 10 mM CaCl₂); 10 mM NaN₃ and0.01% Triton X-100) containing 1% raw corn starch, or 0.5% dextrin or 1%“soluble corn starch” (see note below on the “soluble starch”preparation). Assays were performed at 0.5 ml scale for glucoamylase and0.25 ml scale for α-amylase in an Eppendorf tabletop incubator withconstant shaking (800 rpm).

For glucoamylases, reactions were started by adding the enzyme (finalconcentration 0.25 μg/ml) to the reaction mix. At 0, 2.5, 5, 7.5, 10,15, 20 and 30 min, 50 μl aliquots of the reactions were withdrawn andquenched by addition to 100 μl of 1M Tris buffer, pH 7.5.

For α-amylases, reactions were started by adding the enzyme (finalconcentration 0.4 μg total protein/ml for SEQ ID NO:56, SEQ ID NO:2 andSEQ ID NO:52; 2 μg/ml for SEQ ID NO:62; 4 μg/ml for SEQ ID NO:70 and SEQID NO:66) to the reaction mix, and 10 μl aliquots of reactions werewithdrawn and quenched in BCA reagent at 2, 5, 10, 15, 20, 25, 30, and40 min.

For determination of temperature profiles, assays were performed at 30,34, 37 and 40° C.

The effect of pH on glucoamylase and amylase activities was evaluated atpH 3.5, 4, 5, 6 and 7, using the broad pH range Britton-Robinson buffer(50 mM CH3COOH; H₃PO₄, H₃BO₃). Parallel reactions at pH 4, 5 and 6 werealso performed in the presence of 50 mM acetate buffer to ensure thatthe buffer used did not influence the results. For the determination ofpH profile of two calcium-dependent α-amylases (SEQ ID NO:56 and SEQ IDNO:62), malic acid/acetate/IVIES buffers were used instead ofBritton-Robinson.

Preparation of “soluble corn starch” for reaction with α-amylases.Dextrin (Sigma D2006) could not be used as a substrate in the BCAα-amylase reactions due to the high reducing ends background. Thereforea heated corn starch was employed as a substrate. Specifically, 2% cornstarch was dissolved in deionized water and heated with mixing in aboiling water bath for 30-40 minutes, until the starch had dissolved andthe solution appeared milky, but translucent. The solution of heatedstarch was used for 2 days, after which time some signs ofretrogradation were observed (appearance of starch clumps), and thesolution was discarded.

8. Glucose Oxidase/Peroxidase (GO) Assay for the Quantification ofGlucose Released During Starch Hydrolysis.

A coupled glucose oxidase/peroxidase (GO) assay was used to determinethe amount of glucose released by glucoamylase during starch hydrolysis.GO reactions were started by adding 10 μl of the quenched starchhydrolysis reaction to 90 μl of PBS containing glucose oxidase (0.1U/ml), peroxidase (0.25 U/ml) and 0.05 mM Amplex Red, in black Nunc96-well plates. Plates were kept at room temperature in the dark for 30min prior to reading on a fluorescent plate reader with Ex/Em 545/590nm. A standard curve with glucose concentrations of 0 to 100 μM was usedto assess the amount of glucose produced in the hydrolysis reactions.Initial rates of starch hydrolysis (nmols of glucose released from 1%granular starch/min/μg glucoamylase) were determined by plotting theamount of glucose released over time, and calculating the slope of thebest linear fit through the data points.

9. BCA Assay for Determining the Increase in Concentration of ReducingEnds During Starch Hydrolysis.

A 10 μl aliquot of amylase starch hydrolysis reaction was quenched into100 μl of BCA reagent (consisting of 64 mg/mL sodium carbonatemonohydrate, 24 mg/mL sodium bicarbonate, 1.95 mg/mL BCA, 1.24 mg/mLcupric sulfate pentahydrate, 1.26 mg/mL L-serine). Color developmentoccurred during incubation of the quenched reaction at 80° C. for 35minutes and was followed by absorbance determination at 560 nm. Initialrates were calculated over a 40 min reaction time. A standard curveusing maltose (0-54 μM) was constructed to correlate A_(560 nm) with theconcentration of generated reducing sugars (nmoles). Specific activitywas expressed as nmoles/min/μg enzyme.

10. Bond-Type Specificity of Glucoamylases with Maltose and Isomaltoseas Substrates.

Reactions were started by adding the enzyme (final concentration 5 μg/mlfor maltose, and 30 μg/ml for isomaltose) to the reaction mix. At 2, 5,10, 15, 20, 25, 30 and 40 min, 5 μl aliquots of the reactions werewithdrawn and quenched by addition to 10 μl of 1M Tris buffer, pH 7.5.Nine substrate concentrations were used in the studies, ranging from 0to 12 mM for maltose and 2.5 to 120 mM for isomaltose. The reactionswere performed in triplicate at 37° C. and pH 5.0 in buffer (50 mMNaCH₃CO₂, 10 mM CaCl₂)), at 50 ul scale in an Eppendorf tabletopincubator with constant shaking (800 rpm). Glucose production wasmeasured at the end of the reaction using the glucose oxidase/peroxidase(GO) assay.

Results

2. Characterization of Glucoamylases

1.1 Initial Reaction Rates:

Initial rates for granular and soluble starch hydrolysis are presentedin Table 1. As can be seen from Table 1, exemplary glucoamylases of thisinvention displayed up to 3× better activity (SEQ ID NO:48) againstgranular starch, with similar or slightly better activity on solublestarch when compared to the benchmark A. niger enzyme. SEQ ID NO:48 wasalso expressed in E. coli (see above section on Pichia expressionconstructs)—this E. coli expressed enzyme is labeled SEQ ID NO:48 (Ec)in the table below. SEQ ID NO:48(Ec) did not display any activityagainst granular starch under the conditions tested (probably due to thelack of a Starch Binding Domain).

Table 1: Comparison of initial rates of granular corn starch and solublestarch (dextrin) hydrolysis by “lead” exemplary amylases and/orglucoamylases and a benchmark enzyme A. niger glucoamylase (Sigma A7095)at 37° C., pH 5.0:

TABLE 1 Initial rate* ± SD Initial rate* ± SD Enzyme Granular starchSoluble starch SEQ ID NO: 48 35.6 ± 3.8 60.7 ± 5.1 SEQ ID NO: 26 28.8 ±3.4 51.8 ± 7.4 SEQ ID NO: 74 25.1 ± 2.5 84.3 ± 3.5 SEQ ID NO: 18 24.3 ±4.3 58.3 ± 3.0 SEQ ID NO: 28 17.8 ± 4.3 33.8 ± 4.0 SEQ ID NO: 14  6.6 ±1.2 53.9 ± 4.1 SEQ ID NO: 48(Ec) 0 59.3 ± 8.5 A. niger 11.3 ± 2.7 43.3 ±7.4 glucoamylase (Sigma A7095) *Initial rates are expressed as nmols ofglucose/min/μg of glucoamylase protein released from 1% granular starchor from 0.5% dextrin. Each number is the average value from 6-10 datapoints.

1.2 Temperature Profile:

The effect of temperature (30° C.-40° C.) on starch hydrolysis by thecharacterized glucoamylases is presented in FIGS. 1A and 1B. Activitiesof glucoamylases increased with temperature; they were most active at40° C. but retained ˜50% of peak activity at 30° C.

FIG. 28A: Effect of Temperature on the Activity of exemplary amylasesand/or glucoamylases and the glucoamylase A. niger glucoamylase withGranular Starch as substrate (Benchmark A. niger glucoamylase (SigmaA7095). Glucose release was measured at the indicated temperature at pH5.0. The exemplary SEQ ID NO:20 is not included in the plot because itdid not display any activity against granular starch under theseparticular conditions as tested.

FIG. 28B: Effect of Temperature on the Activity of the exemplaryglucoamylase SEQ ID NO:20 and the glucoamylase A. niger glucoamylase(Sigma A7095) with Soluble Starch (Dextrin) as a substrate. Glucoserelease was measured at the indicated temperature at pH 5.0.

1.3 pH Profile:

The influence of pH on starch hydrolysis was tested with both granularand soluble starch and the results are presented in FIG. 2A and FIG. 2B,respectively. All glucoamylases hydrolyzed both substrates best at lowerpH with SEQ ID NO:26 being the most acidic in character.

FIG. 29A: Effect of pH on the Activity of Glucoamylases with GranularStarch as substrate. Glucose release was measured at the indicated pH at37° C. Initial rates were calculated over 20 min and converted to apercentage of the maximum rate. The exemplary SEQ ID NO:20 is notincluded in the plot because it did not display any activity againstgranular starch, under the particular conditions tested here.

FIG. 29B: Effect of pH on the Activity of Glucoamylases with SolubleStarch as substrate. Glucose released was measured at the indicated pHat 37° C. Initial rates were calculated over 20 min and converted to apercentage of the maximum rate.

1.4 Bond-Type Cleavage Specificity:

The kinetic parameters for the hydrolysis of maltose (alpha-1,4-linkage)(maltose is two alpha-D-glucoses) and isomaltose (alpha-1,6-linkage)were determined for 7 selected glucoamylases and the benchmark (A. nigerglucoamylase (Sigma A7095). The experiments were conducted withlyophilized P. pastoris lysates and the proteins were not purified;therefore only data independent of protein concentration are reported inthis document. Table 2, below, summarizes values of K_(M) for maltoseand isomaltose and the ratio of k_(cat)/K_(M) for maltose compared tok_(cat)/K_(M) for isomaltose. These parameters determined for A. nigerglucoamylase (Sigma A7095) are in good agreement with published data(K_(M) for maltose is reported to be 1.2-2.1 mM; K_(M) for isomaltose isreported to be 19.8-42.0 and k_(cat)/K_(M) for maltose overk_(cat)/K_(M) for isomaltose is reported to be between 300-600 accordingto Frandesen at al. 1995; Sierks and Svensson; 1996; Fagerstrom andKalkkinen; 1995).

As can be seen from Table 2, the exemplary glucoamylase SEQ ID NO:20 wasmost strongly selective for maltose and had nearly 900-fold higherspecificity towards alpha-1,4-linkages relative to alpha-1,6-bonds. Theleast selective glucoamylase was SEQ ID NO:14 with ˜100-fold higherspecificity towards alpha-1,4-bonds relative to alpha-1,6-bonds.

TABLE 2 Kinetic parameters for hydrolysis of Maltose and Isomaltose by 7exemplary glucoamylases of this invention and a benchmark (A. nigerglucoamylase (Sigma A7095)). Maltose Isomaltose k_(cat)/K_(M) (maltose)/Enzyme K_(M) (mM) K_(M) (mM) k_(cat)/K_(M) (isomaltose) SEQ ID NO: 280.61 ± 0.06 11.94 ± 4.99 750 SEQ ID NO: 74 1.87 ± 0.17 11.55 ± 2.92 481SEQ ID NO: 20 2.62 ± 0.19  53.97 ± 23.17 897 SEQ ID NO: 14 2.67 ± 0.15 41.5 ± 5.05 116 SEQ ID NO: 26 0.98 ± 0.33 12.18 ± 0.64 456 SEQ ID NO:48 2.26 ± 0.12 21.69 ± 3   565 SEQ ID NO: 18 1.01 ± 0.09 11.74 ± 7.74415 A. niger 0.93 ± 0.1  18.72 ± 3.95 249 glucoamylase (Sigma A7095)Each number is the average value from 5 different experiments.

REFERENCES

-   1. Frandsen T P, Christensen T, Stoffer B, Lehmbeck J, Dupont C,    Honzatko R B, Svensson B (1995) Biochemistry. 34:10162-9.-   2. Sierks M R and Svensson B. (1996) Biochemistry; 35:1865-71.-   3. Fagerstrom R and Kalkkinen N. (1995) Biotechnol Appl Biochem.    21:223-31.    Characterization of Amylases

Initial Reaction Rates:

The initial rates of hydrolysis of granular and soluble starch arepresented in Table 3. Eight exemplary alpha-amylases were compared witha benchmark alpha-amylase from A. oryzae. As can be seen from Table 3,below all amylases tested displayed significantly higher activityagainst soluble starch when compared with granular starch. However, thisdifference was less marked for Amylases and/or glucoamylases than forthe benchmark enzyme.

Table 3: Comparison of initial rates of hydrolysis of granular cornstarch and soluble corn starch by 8 α-amylases and a benchmarkalpha-amylase from A. oryzae at 37° C. and pH 5:

TABLE 3 Initial rate* ± SD Initial rate* ± SD Exemplary Enzyme Granularstarch Soluble starch SEQ ID NO: 56 15.7 ± 1.67 1607.9 ± 518.22 SEQ IDNO: 70** 20.5 109.1 SEQ ID NO: 62  3.5 ± 0.37 139.6 ± 55.96 SEQ ID NO:66**  2.1  70.8 SEQ ID NO: 2   7 ± 0.75 381.2 ± 74.15 SEQ ID NO: 52 10.3± 1.75 248.2 ± 28.46 SEQ ID NO: 78  0.4 ± 0.06   232 ± 52.63 SEQ ID NO:76** 25.2 809.1 A. oryzae amylase  0.4 ± 0.07 498.7 ± 64.78(MegazymeE-ANAAM) *Initial rates are expressed as nmole of reducing endsreleased from 1% starch/min/μg of alpha-amylase protein. **Data obtainedusing purified enzyme. Each number is an average value from 5 datapoints.

Temperature Profile:

The effect of temperature on starch hydrolysis by the characterizedα-amylases is presented in FIG. 30. Activities of amylases were affectedby temperature to a different degree. Five of the exemplary amylases ofthis invention (SEQ ID NO:56, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:76and SEQ ID NO:66) were most active at 40° C. and retained ˜30% ofactivity at 30° C. Activities of the exemplary SEQ ID NO:2, SEQ ID NO:52and SEQ ID NO:62 were only marginally affected by changes in temperatureover the range investigated.

FIG. 30 illustrates: the Influence of Temperature on Starch Hydrolysisby 9 α-amylases. Activity was measured at pH 5.0 during 40 minincubation at the indicated temperature, and initial rates werecalculated and plotted against time. In FIG. 30:

-   -   Rates for the exemplary SEQ ID NO:56, SEQ ID NO:2 and SEQ ID        NO:52 are presented on the left axis; rates for the rest of        enzymes are presented on the right axis.    -   Initial rates are expressed as nmoles of reducing ends released        from 1% granular corn starch/min/μg of enzyme in 250 μl        reaction.    -   Activities of the exemplary SEQ ID NO:70, SEQ ID NO:66 and SEQ        ID NO:76 are expressed in nmoles/min/ng of total protein in        Pichia pastoris supernatant.

pH Profile:

The influence of pH on starch hydrolysis was tested with both granularand soluble starch substrates and the results are presented in FIG. 31Aand FIG. 31B, respectively. The exemplary SEQ ID NO: 52 (of fungalorigin) had the lowest pH optimum (˜pH 4). Another exemplary enzyme (SEQID NO:2) (of fungal origin) also displayed preference for acidic pH,with an apparent optimum at ˜pH 4.5-5. The exemplary amylase SEQ IDNO:66 (of Archaeal origin) had an apparent optimum of ˜pH 5.0, retaining˜70% of peak activity at pH 4.0. The remaining enzymes had apparentoptima between pH 5.0 and 6.0, and were almost inactive at pH 4.0 and3.5.

FIG. 31A: Effect of pH on the activities of alpha-amylases with GranularStarch as substrate. The increase in reducing ends was measured at theindicated pH at 37° C. Initial rates were calculated over 40 min andconverted to a percentage of the maximum rate.

FIG. 31B: Effect of pH on the activities of alpha-amylases with SolubleStarch as substrate. The increase in reducing ends was measured at theindicated pH at 37° C. Initial rates were calculated over 40 min andconverted to a percentage of the maximum rate.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made without departing fromthe spirit and scope. Accordingly, other embodiments are within thescope of the following claims.

What is claimed is:
 1. A method for hydrolyzing starch comprising: (a)providing a starch; (b) providing a composition comprising a blend of apolypeptide having amylase activity and a polypeptide havingglucoamylase activity; (c) contacting the starch of (a) with thecomposition of (b), wherein the polypeptides hydrolyze the starch;wherein the polypeptide having amylase activity has an amino acidsequence having at least 85% sequence identity to the amino acidsequence of SEQ ID NO: 52; and the polypeptide having glucoamylaseactivity has an amino acid sequence having at least 85% sequenceidentity to the amino acid sequence of SEQ ID NO:28.
 2. The method ofclaim 1, wherein the starch is from rice, corn, barley, wheat, legumes,potato, sweet potato, a fermentable sugar, or any combination thereof.3. The method of claim 1, wherein the starch is an oligosaccharide. 4.The method of claim 1, wherein the starch is a polysaccharide.
 5. Themethod of claim 1, wherein the starch is liquified.
 6. The method ofclaim 1, wherein the polypeptide is thermostable and retains the amylaseor glucoamylase activity under conditions comprising a temperature rangefrom 37° C. to 95° C.
 7. The method of claim 1, wherein the compositionis selected from the group of: a food, a feed, a beverage, a detergent,a textile, a fabric, a paper, a paper product, a paper pulp, a liquid,and a syrup.
 8. The method of claim 1, wherein the hydrolyzing of starchis during a liquefaction stage.
 9. The method of claim 1, wherein thehydrolyzing of starch is during a saccharification stage.
 10. The methodof claim 1, wherein the hydrolyzing of starch is during a fermentationstage.
 11. The method of claim 1, wherein the hydrolyzing of starch isduring a simultaneous liquefaction, saccharification, and fermentationstage.
 12. The method of claim 1, wherein the hydrolyzing of starch isduring a brewing stage.